Attenuated Salmonella SP12 mutants as antigen carriers

ABSTRACT

The present invention relates to vaccines, in particular, to an attenuated gram-negative cell comprising the SPI2 gene locus, wherein at least one gene of the SPI2 locus is inactivated, wherein said inactivation results in an attenuation/reduction of virulence compared to the wild type of said cell, and to a carrier for the presentation of an antigen to a host, which carrier is said attenuated gram-negative cell, wherein said cell comprises at least one heterologous nucleic acid molecule comprising a nucleic acid sequence coding for said antigen, wherein said cell is capable of expressing said nucleic acid molecule or capable of causing the expression of said nucleic acid molecule in a target cell.

This application is a divisional of U.S. Ser. No. 09/763,620 filed Mar.2, 2001, now U.S. Pat. No. 6,936,425 issued Aug. 30, 2005, entitled“Attenuated Salmonella SP 12 Mutants as Antigen Carriers”, which is afiling under 35 USC §371 of PCT/EP99/06514 (WO 00/14240) filed on Sep.3, 1999, which claims priority to European Patent Application No.98116827.1 filed Sep. 4, 1998.

BACKGROUND OF THE INVENTION

In 1996, over 17 million people world-wide, mainly in developingcountries, were killed by various infections. The appearance and spreadof antibiotic resistances coupled with the increase in world-wide travelhas led to an increasing risk for the outbreak of pandemic infections.This possibility must be taken very seriously since, for some pathogenicbacteria, the therapeutic alternatives available have been reduced to asingle option. Intriguingly, pathogenic bacteria have also beendiscovered to be a relevant factor in many chronic diseases. Stomachcancer, for example, is the second most common cancer world-wide and isdirectly linked with chronic Helicobacter pylori infections. Chlamydiapneumoniae has been detected in arteriosclerotic plaques and recentlythis bacterium has been found in the diseased regions of the brain ofpeople suffering from Alzheimer's disease. Many autoimmune diseases,such as rheumatoid arthritis, seem to have bacterial origin. Borreliaburgdorferi is, in addition to many other bacteria, a prominent exampleof an organism causing disease affecting increasing numbers of people.Finally, Nanobacteria have been identified in the chronically diseasedkidneys of patients with crystalline deposits. Other serious chronicdiseases are caused by viral pathogens, the most clinically relevant areHepatitis B and C viruses (liver cancer) and the human papilloma virus(cervical cancer).

The increasing clinical importance of bacterial pathogens has provokedincreased discussion regarding the paradigm of medicinal treatment orprevention as the means to handle chronic diseases. Consistently, somechronic diseases have been successfully cured by antibiotic treatment.However, as indicated above, all micro-organisms are genetically capableof rapidly generating progenies with adequate antibiotic resistances,thus impeding efficient routine treatment. Conclusively, vaccinesrepresent an excellent alternative to pharmacological drugs, and,considering the financial aspect that disease prevention is lesscost-intensive than therapy, the option of vaccination is even moreattractive. Therefore, the therapeutic vaccination approach has becomeparticularly relevant, especially with respect to the treatment ofcancer and chronic bacterial or viral diseases.

The most frequently practised approach uses oral delivery of eitherinactivated pathogens (dead vaccine) or parenteral injections of adefined mixture of purified components (subunit vaccines). Most of thedead vaccines are efficacious, however, the risk that the inactivationprocedure was incomplete and that the vaccinee may become infectedremains a problem. Furthermore, dead vaccines very often do not coverall genetic variants that appear in nature. The subunit vaccines abolishmost of the disadvantages of the traditional dead vaccines. However,they require technologically advanced antigen and adjuvant preparations,which makes such vaccines relatively expensive. Furthermore, the subunitvaccines are preferentially inoculated by the parenteral route, which isnot the optimal route for eliciting a broad immune response. Inparticular, the mucosal branch of the immune system, which is theprimary line of protection against many pathogens, is strongly neglectedby parenteral immunisations.

Another generation of vaccines is represented by live attenuatedvaccines, which are based on pathogenic bacteria or viruses that havebeen mutated to apathogenic variants. These variants multiply in vivofor a limited period of time before they are completely cleared by thehost. Their limited prevalence in the host tissue is sufficient toadequately provoke the host immune system, which is then able toestablish a protective immune response. From the safety aspect, liveattenuated bacterial vaccines are more favoured than live attenuatedviral vaccines. Should a live bacterial vaccine becomes threating for avaccinee, the attenuated bacteria can generally be controlled byantibiotic treatment. In contrast, live viral vaccines, which use thereplication apparatus of the host cell, are almost impossible tocontrol. Live bacterial vaccines are typically administered orally andserve as excellent stimulators of the mucosal immune system. Moreover,live bacterial vaccines are also good stimulators of the systemicallyactive immune system, namely the humoral and cellular branches. Due tothese excellent immuno-stimulatory characteristics, live bacterialvaccine strains, such as Salmonella, are ideal carriers for expressingantigens from a heterologous pathogen. Such bivalent (or multivalent)vaccines mediate protection against two pathogens: the pathogenhomologous to the carrier as well as the pathogen whose protectiveantigen(s) are expressed by the carrier. Although no bivalent bacterialvaccine expressing heterologous antigens is currently in use, potentialcarriers currently under investigation include Bacille Calmette-Guerin(BCG), Salmonella species, Vibrio cholerae and Escherichia coli.

In the attenuation process, mutations are preferentially targeted togenes that support the survival of the pathogen in the host. Initially,chemical mutation regimes were applied to the Salmonella typhi strainTy2, resulting in what were thought to be perfectly attenuated pathogenscapable of mediating protective immunity, in contrast to the deadhomologue. However, subsequent large-scale clinical trails revealed thatsuch strains were still not sufficiently efficacious in the preventionof typhoid fever. It appears that such strains were mutated in severalgenes, resulting in an over-attenuation, which adversely affects theimmunogenic potential of the strain. Novel typhoid vaccine strains havebeen developed by the introduction of genetically defined mutations.Most of these mutations have been established in S. typhimurium.Infection with S. typhimurium causes typhoid fever-like symptoms in miceand murine salmonellosis is a well accepted model for human typhoid.Such vaccine strains contain mutations in proteins causing deficienciesin the biosynthesis of aromatic amino acids (e.g. aroA, aroC and aroD)or purines (e.g. purA and purE), in the adenylate cyclase gene (cya) orthe cAMP receptor protein (crp), or possess mutations affecting theregulation of several virulence factors (phoP and phoQ). However,although a number of attenuated mutants have been generated andcharacterised in the mouse model with regard to their role in virulence,relatively few of them have been evaluated as vaccine carriers inhumans. The reason for this is that the mutants used are either stilltoo virulent, causing severe side effects in the host, or are notsufficiently immunogenic, due to inadequate presentation to the immunesystem, which requires a critical level of persistence of the vaccinestrain in the host for activation.

A recent study revealed that the inactivation of individual Salmonellagenes causing attenuation of virulence directly influences the qualityof an immune response against the vaccine carrier strain. From thisfinding, one can conclude that it might be possible to generate avariety of differently attenuated Salmonella vaccine strains, each witha unique profile and individual capabilities for eliciting an immuneresponse. With this repertoire, it might be possible to tailor a vaccinestrain according to specific immunological demands. As a logicalconsequence, one should also be able to develop attenuated Salmonellavaccine strains for either prophylactic or therapeutic purposes.However, the means by which such a representative repertoire ofSalmonella vaccine strains is obtained and further developed into anefficacious vaccine must be determined.

In cases in which a Salmonella vaccine strain is used as a carrier forheterologous antigens, additional parameters must be considered.Traditionally, heterologous antigens have been expressed in theSalmonella cytosol. In the mouse typhoid model, it was demonstratedthat, when heterologous antigens are expressed at high levels in theSalmonella cytosol, inclusion bodies are often formed, which negativelyinfluence the immunogenicity of the recombinant live vaccine strain inthe vaccinated host. It was concluded that the formation of inclusionbodies might be fatal for the bacterium, further decreasing vitality andincreasing attenuation, and thus lowering the immunogenicity. Indeed,specific expression systems that circumvent this secondary attenuationprinciple, e.g. the 2-phase regulated expression system, can improve theefficacy of the presentation of heterologous antigens to the host immunesystem.

It has been demonstrated that secretion of antigens by live attenuatedSalmonella can be superior to intracellular expression of the sameantigens both in eliciting protective T-cell responses (Hess et al.,1996; Hess et al., 1997b) and in eliciting elevated levels ofantigen-specific antibody (Gentschev et al., 1998). Efficiencies ofHlyA-directed secretion systems, however, are usually low (30% or lessof total synthesized antigen) (Hess et al., 1997a; Hess et al., 1996),and the system seems to be problematical in S. typhi for export ofheterologous antigens (Orr et al., 1999).

A similar immunological profile is induced by the two type III secretionsystems, which are encoded by the Salmonella Pathogenicity Islands 1 and2. These complex secretion machineries naturally deliver “effectorproteins” into the cytosol of the infected host cell, supporting thesurvival of the pathogen within the host cell. By means of genetechnology, the “effector proteins” can be converted into carriervehicles for epitopes from heterologous antigens. Such chimeric“effector proteins” lose their virulent character but retain theirsecretory character. Consequently, the chimeric “effector protein” isdelivered into the lumen of the host cell, where it is appropriatelyprocessed and subsequently stimulates the cytotoxic branch of the hostimmune system.

The most abundant protein secreted by Salmonella is flagellin (see, forexample (Hueck et al., 1995)). In S. typhimurium, flagellin occurs intwo allelic variants, FliC or FljB, while S. typhi carries only the FliCgene. Flagellin is secreted via the flagellum-specific export (FEX)pathway (Macnab, 1996; Minamino and Macnab, 1999), which is homologousto the type III secretion pathway (Hueck, 1998). It also has been shownrecently that the FEX pathway functions in secretion of non-flagellaproteins in Yersinia enterocolitica (Young et al., 1999). Like in typeIII secretion, the amino terminus of FliC directs secretion. Thus, atruncated version of 183 amino terminal amino acids of FliC (full lengthis 495 aa) is constitutively secreted in large amounts (Kuwajima et al.,1989). In analogy to type III secretion, the effective secretion signalin FliC may be as short as 10 to 20 amino acids. The FliC or FljBsecretion signals can potentially be used to secrete large quantities ofa heterologous protein which can serve as an antigen in heterologousvaccination. It is likely that the amount of secreted antigen can beeven further increased in regulatory mutants affecting the expression offlagella biosynthesis genes (Macnab, 1996; Schmitt et al., 1996) or byusing recombinant promoters to drive expression of the flagellin gene.

Secretion via the FEX pathway can allow the delivery of large amounts ofantigen into the Salmonella-containing phagosome for early and efficientantigen processing and antigen presentation to the host immune system.Especially the MHC class II dependent branch of the host immune systemis strongly supported by the FEX pathway mediated antigen delivery.

The other known export machineries and surface display systems ofGram-negative bacteria can be also applied to bacterial vaccine carrierssuch as Salmonella. In general, a good immune response is achieved whenthe antigen is presented on the Salmonella surface. However, as littleis known about the immunological consequence of such antigenpresentation systems, further experimental work is needed.

Additional immuno-modulatory effects can be achieved whenenvironmentally regulated Salmonella promoters are used for theexpression of heterologous antigens. For instance, the expression of aheterologous gene in a Salmonella carrier strain under control of the invivo regulated stress response htrA gene promoter resulted in a strongerimmune response than was obtained when under control of theanaerobically inducible promoter of the nirB gene.

According to a first aspect, the present invention relates to anisolated nucleic acid molecule comprising a nucleic acid sequencecomprising at least 50 nucleotides a) of the nucleic acid sequence ofone of FIGS. 21A, B, b) of an allele of the nucleic acid sequence of oneof FIGS. 21A, B or c) of a nucleic acid sequence which under stringentconditions hybridizes with the nucleic acid sequence of one of FIGS.21A, B.

Stringent hybridization conditions in the sense of the present inventionare defined as those described by Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press (1989),1.101-1.104. According to this, hybridization under stringent conditionsmeans that a positive hybridization signal is still observed afterwashing for 1 hour with 1×SSC buffer and 0.1% SDS at 55° C., preferablyat 62° C. and most preferably at 68° C., in particular, for 1 hour in0.2×SSC buffer and 0.1% SDS at 55° C., preferably at 62° C. and mostpreferably at 68° C.

In particular, the present invention relates to such a nucleic acidmolecule which comprises the complete coding regions or parts thereof ofthe genes ssaD, ssaE, sseA, sseB, sscA, sseC, sseD, sseE, sscB, sseF,sseG, ssaG, ssaH, ssaE, ssaJ, ssrA and ssrB. The invention pertains alsoto such nucleic acids, wherein at least one coding region of said genesis functionally deleted.

In one embodiment, the nucleic acid molecule comprises an insertioncassette to facilitate the insertion of a heterologous nucleic acidmolecule by transposon or phage mediated mechanism.

Furthermore, said nucleic acid molecules can comprise at least oneheterologous nucleic acid molecule. In this case the heterologousnucleic acid molecule may be fused 5′ or 3′, inserted ordeletion-inserted to the inventive nucleic acid molecule. By the term“deletion-inserted” it is understood that the insertion of theheterologous nucleic acid molecule is associated with a concurrentdeletion of parts of the inventive nucleic acid molecule. Preferably,the nucleic acid molecule is inserted or deletion-inserted and in onepreferred embodiment the heterologous nucleic acid molecule is flanked5′ and 3′ by sequences of the nucleic acid molecule according to theinvention, wherein each of said sequences has a length of at least 50nucleotides, preferably 200-250 nucleotides.

Preferred, the heterologous nucleic acid molecule codes for apolypeptide or peptide, more preferred it codes for a bacterial or viralantigen or a homologue thereof or for a tumor antigen.

It is preferred that the nucleic acid molecule also comprises at leastone gene expression cassette to allow for efficient expression of theheterologous nucleic acid molecule. Such gene expression cassetteusually comprises elements such as promoters and/or enhancers whichimprove the expression of the heterologous nucleic molecule acids.Usually, such gene expression cassette comprises elements for thetermination of transcription. The presence of transcription terminators,however, may be not preferred in cases where the heterologous nucleicacid molecule is to be transcribed together with other genes into acistronic mRNA.

The nucleic acid molecule, one or more selective marker cassettes andone or more transactivator cassettes and optionally invertase cassettesfor allowing the expression of the heterologous nucleic acid moleculesin a one-phase system or a two-phase system. Furthermore, sequences maybe present which code for a polypeptide or peptide-targeting domain and,thus, allow for the targeting of the expression product of theheterologous nucleic acid molecule to a predetermined cell compartmentsuch as cytosol, periplasma or outer membrane, or the secretion of saidexpression product, or which code for an immunostimulatory domain.

According to another aspect, the invention relates to a recombinantvector which comprises the nucleic acid molecule described above.Another aspect of the invention pertains to a cell comprising a modifiedinventive nucleic acid molecule as described above by insertion of aheterologous sequence or the recombinant vector. The cell may be aprokaryotic cell such as a gram-negative cell, e.g. a Salmonella cell,or it can be a eukaryotic cell such as a mammalian cell, e.g. a humancell, and, in particular, a macrophage.

According to a still further aspect, the present invention relates to apeptide or polypeptide comprising a peptide sequence comprising at least20 amino acids a) of the sequence of one of FIGS. 23A-Q , or b) of asequence which is 60%, preferred 65% and more preferred 70% homologousto the sequence of one of FIGS. 23A-Q. In particular, the inventionrelates to a polypeptide comprising the sequence a) of one of FIGS.23A-Q, or b) which is 60%, preferred 65% and more preferred 70%homologous to the sequence of one of FIGS. 23A-Q.

Percent (%) homology are determined according to the following equation:

$H = {\frac{n}{L} \times 100}$wherein H are % homology, L is the length of the basic sequence and n isthe number of nucleotide or amino acid differences of a sequence to thegiven basic sequence.

Another aspect of the present invention relates to an antibody which isdirected against an epitope which is comprised of the aforementionedpeptide or polypeptide. The antibody may be polyclonal or monoclonal.Methods for producing such an antibody are known to the person skilledin the art.

A further aspect of the present invention relates to a fusion proteincomprising the polypeptide according to any one of the claims 17 and 18having inserted or deletion-inserted or being fused C- or NH₂-terminallywith at least one heterologous polypeptide. The heterologous polypeptidepreferred is an antigen, more preferred a bacterial or viral antigen ora tumor antigen.

The present invention furthermore provides instructions for thedevelopment of a variety of potential live Salmonella vaccine strainswith different attenuation levels, which subsequently serve as platformsfor the development of recombinant live Salmonella vaccine carrierstrains that express antigens from heterologous pathogens, thus servingas multivalent vaccines. Such recombinant live Salmonella vaccinecarriers are equipped with modules comprising variable gene cassettesthat regulate the expression of heterologous antigens in Salmonella anddetermine presentation of the heterologous antigens to the host immunesystem. By combinations of both systems, differently attenuated liveSalmonella vaccine strains and variable gene cassettes, a variety ofrecombinant live vaccine carrier strains can be generated that have, dueto their variable immunogenic characteristics, a broad applicationspectrum for both prophylactic and therapeutic use. The basicattenuation principle originates from novel mutations in the SalmonellaPathogenicity Island 2 (SPI2) gene locus. Additional mutations, whichcan be used either alone or in combination with mutations in sse orSPI-2 genes or in combination with the aroA mutation for optimalattenuation of live vaccine carrier strains, have been reported recently(Heithoff et al., 1999; Valentine et al., 1998). By combination of theindividual mutations in the SPI-2 gene locus with each other and withother known attenuating gene mutations, such as aroA, etc., a broadrepertoire of attenuation and immunogenicity can be achieved. Differentexpression cassettes can be introduced on these platforms, allowingfurther modulation of the immune response directed against theheterologous antigens. Finally, a library of individual recombinant liveSalmonella vaccine carrier strains is generated, covering a broadspectrum of immuno-stimulatory potential, from which a genuine livevaccine strain can be tailored for the optimal protection or treatmentof humans and/or animals against specific pathogens or disease.

Thus, in a further aspect, the present invention is an attenuatedgram-negative cell comprising the SPI2 gene locus, wherein at least onegene of the SPI2 locus is inactivated, wherein said inactivation resultsin an attenuation/reduction of virulence compared to the wild type ofsaid cell.

Genes present in the Salmonella pathogenicity island 2 that encode for avariety of proteins involved in type III secretion and those that arerequired for systemic spread and survival within phagocytic cells areideal candidates for attenuation of pathogenic Salmonella ssp.

Several gram-negative bacterial pathogens secrete certain virulenceproteins via specialised type III secretion systems. Virulence factorsenable pathogenic bacteria to colonise a niche in the host despitespecific attacks of the immune system. The type III secretion systemscomprise a large number of proteins required to transfer specificeffector proteins into eukaryotic host cells in a contact-dependentmanner, thus they have also been called contact-dependent secretorysystems. Although several components of the secretion system apparatusshow evolutionary and functional conservation across bacterial species,the effector proteins are less well conserved and have differentfunctions. The Yersinia effectors YpkA and YopH have threoninelserinekinase and tyrosine phosphatase activities, respectively. The actions ofthese and other Yops inhibit bacterial phagocytosis by host cells, whichis thought to enable extracellular bacterial proliferation. The ShigellaIpa proteins, secreted by the mxi/spa type III secretion system, promoteentry of this bacterium into epithelial cells. EspA, EspB and EspD,encoded by the locus of enterocyte effacement (LEE) of enteropathogenicEscherichia coli (EPEC) are required for translocation of proteins thatcause cytoskeletal rearrangements and the formation of pedestal-likestructures on the host cell surface.

For the purposes of the present invention an “gram-negative cellcomprising the SPI2 gene locus” is a cell having a gene locus thatharbors genes required for the systemic spread and survival withinphagocytic cells and, thus, is a homologue or functional equivalent ofthe SPI2 locus from Salmonella. Preferred, the inventive attenuatedgram-negative cell is an Enterobactericae cell, more preferred, aSalmonella cell, a Shigella cell or a Vibrio cell. In general, cellshaving a broad host range are preferred. Typical hosts are mammals, e.g.man, and birds, e.g. chicken. Salmonella cells are more preferred, andparticularly preferred is Salmonella serotype typhimurium DefinitiveType 104 (DT 104).

Salmonella typhimurium is unusual in that it contains two type IIIsecretion systems for virulence determinants. The first controlsbacterial invasion of epithelial cells, and is encoded by genes within a40 kb pathogenicity island (SPI1). The other is encoded by genes withina second 40 kb pathogenicity island (SPI2) and is required for systemicgrowth of this pathogen within its host. The genes located onpathogenicity island SPI1 are mainly responsible for early steps of theinfection process, the invasion of non-phagocytic host cells by thebacterium. For most of the SPI1 genes, mutations result in a reducedinvasiveness in vitro. However, mutants that are defective in invasionare not necessarily avirulent; studies in mice demonstrated that, whilethese mutations in SPI1 genes significantly reduced virulence upondelivery by the oral route, they had no influence on virulence followingan intraperitoneal route of infection. Taken together, these resultsindicate that mutations in genes within the pathogenicity island SPI1 donot abolish systemic infection and are therefore not very useful for thedevelopment of a safe, attenuated Salmonella carrier strain. Incomparison, virulence studies of SPI2 mutants have shown them to beattenuated by at least five orders of magnitude compared with thewild-type strain after both oral and intraperitoneal inoculation ofmice.

Many of the genes encoding components of the SPI2 secretion system arelocated in a 25 kb segment of SPI2. SPI2 contains genes for a type IIIsecretion apparatus (ssa) and a two component regulatory system (ssr),as well as candidate genes for a set of secreted effectors (sse) andtheir specific chaperones (ssc). On the basis of similarities with genespresent in other bacterial pathogens, the first 13 genes within thessaK/U operon and ssaJ encode components of the secretion systemapparatus. A number of additional genes, including ssaC (orf 11 in Sheaet al., 1996; spiA in Ochman et al., 1996) and ssrA (orf 12 in Shea etal., 1996; spiR in Ochman et al., 1996), which encode a secretion systemapparatus protein and a two component regulatory protein, respectively,are found in a region approximately 8 kb from ssaJ.

Preferably, the inventive attenuated gram-negative cell has inactivatedat least one gene selected from effector (sse) gene secretion apparatus(ssa) genes, chaperon (ssc) genes and regulation (ssr) genes. Morepreferably, the at least one inactivated gene is an sse, ssc and/or ssrgene, even more preferred is an sse and/or ssc gene.

As far as the sse genes are affected by the inactivation, theinactivated gene is preferably sseC, sseD, sseE or a combinationthereof. As far as the ssr genes are affected by the inactivation,preferably at least ssrB is inactivated. As far as the ssc genes areaffected by the inactivation, preferably at least sscB is inactivated.

The inactivation of said gene of the SPI2 locus (or functional homologuethereof in cells other than Salmonella) is effected by a mutation whichmay comprise deletion. Preferred are deletions of at least sixnucleotides, and more preferred is a deletion of the partial and, inparticular, the complete coding sequence for said gene. The mutation mayalso comprise the insertion of a heterologous nucleic acid molecule intosaid gene to be inactivated or a combination of deletion and insertion.

Pathogenic Salmonella ssp. serve a basis for the construction of a panelof different live Salmonella vaccine prototypes generated by gradualattenuations accomplished through the introduction of defined SPI2 genelocus mutations. Each resulting individual live Salmonella vaccineprototype is further transformed into a multivalent recombinant vaccineby the introduction of exchangeable DNA modules carrying (1) geneticallyengineered genes from heterologous pathogens and (2) adequate expressionsystems executing efficacious antigen presentation to the host immunesystem. In concert, these features elicit a specific immune responsethat either protects vaccinated hosts against subsequently invadingSalmonella and/or other pathogens (prophylactic vaccination) oreliminates persistant pathogens, such as Helicobacter pylori(therapeutic vaccination).

Pathogenic Salmonella ssp. are gradually attenuated by mutations inindividual virulence genes that are part of the SPI2 gene locus, e.g. ansse gene coding for an effector protein, such as sseC, ssed or sseE, oran ssc gene, such as sscB, coding for a chaperone, or an ssr gene, suchas ssrB, coding for a regulator. Individual mutation of each of thesegenes leads to a unique individual grade of attenuation, which, in turn,effects a characteristic immune response at the mucosal, humoral andcellular levels. The individual grade of attenuation can be moderatelyincreased by combinations of at least two gene mutations within the SPI2gene locus or by combination with a mutation in another Salmonella geneknown to attenuate virulence, e.g. an aro gene, such as aroA. A strongergrade of attenuation is achieved by mutation of a virulence gene that ispart of a polycistronic gene cluster encoding several virulence factors,such as the transcriptional unit comprising the sseC, sseD, sseE andsscB genes, such that the mutation exerts a polar effect, disruptingexpression of the following genes. The grade of attenuation may directlydepend on the number of virulence genes that are affected by the polarmutation as well as their individual characteristics. Finally, thestrongest attenuation is achieved when regulatory genes, such as ssrB,are mutated. Again, each mode of attenuation of a Salmonella ssp. leadsto the generation of a live Salmonella vaccine strain that evokes animmune response at the mucosal, humoral and cellular levels that ischaracteristic for the type and/or combination of attenuating mutationspresent in that strain. The panel of differently attenuated liveSalmonella vaccine strains that is generated represents a pool ofpotential carrier strains from which that carrier can be selected thatprovokes the most efficacious immune response for either the preventionor eradiction of disease in conjunction with the heterologous antigensthat are expressed.

Mutations leading to attenuation of the indicated Salmonella virulencegenes are preferentially introduced by recombinant DNA technology asdefined deletions that either completely delete the selected virulencegene or result in a truncated gene encoding an inactive virulencefactor. In both cases, the mutation involves a single gene and does notaffect expression of neighbouring genes (non-polar mutation). Aninsertional mutation in one of the indicated virulence genes ispreferred when the selected gene is part of a polycistronic virulencegene cluster and all of the following virulence genes are included inthe attenuation process (polar mutation). Insertional mutations withnon-polar effects are in general restricted to genes that are eithersingly transcribed or are localised at the end of a polycistroniccluster, such as ssrB. However, other attenuating mutations can arisespontaneously, by chemical, energy or other forms of physicalmutagenesis or as a result of mating or other forms of genetic exchange.

Thus, the mutation which results in the preparation of the inventiveattenuated gram-negative cell may be a polar or non-polar mutation.Furthermore, the grade of attenuation may be modified by inactivating anadditional gene outside of the SPI2 locus, for example, anothervirulence gene or a gene that is involved in the biosynthesis of ametabolite or a precursor thereof such as the aro genes, in particular,aroA, or any other suitable gene such as superoxide dismutase (SOD).

The attenuated cell according to the invention may furthermore compriseelements which facilitate the detection of said cell and/or theexpression of an inserted heterologous nucleic acid molecule. An exampleof an element which facilitates the detection of the attenuated cell isa selective marker cassette, in particular, a selective marker cassettewhich is capable of conferring antibiotic resistance to the cell. In oneembodiment, the selective marker cassette confers an antiboticresistance for an antibiotic which is not used for therapy in a mammal.Examples of elements which facilitate the expression of a heterologousnucleic acid molecule are a gene expression cassette which may compriseone or more promoter, enhancer, optionally transcription terminator or acombination thereof, a transactivator cassette, an invertase cassettefor 1-phase or 2-phase expression of a heterologous nucleic acid. Anexample of an element which facilitates the insertion of a heterologousnucleic acid molecule is an insertion cassette.

In another aspect, the invention provides a carrier for the presentationof an antigen to a host, which carrier is an attenuated gram-negativecell according to any one of the claims 22 to 49, wherein said cellcomprises at least one heterologous nucleic acid molecule comprising anucleic acid sequence coding for said antigen, wherein said cell iscapable of expressing said nucleid acid molecule or capable of causingthe expression of said nucleic acid molecule in a target cell.

Preferably, said nucleic acid molecules comprises a nucleic acidsequence coding for a bacterial or viral antigen or for a tumor antigen.Examples of bacterial antigens are antigens from Helicobacter pylori,Chlamydia pneumoniae, Borrelia burgdorferi and Nanobacteria. Examples ofviral antigens are antigens from Hepatitis virus, e.g. Hepatitis B andC, human papilloma virus and Herpes virus. The heterologous nucleic acidmolecule may comprise a nucleic acid sequence which codes for at leastone polypeptide or peptide-targeting domain and/or immunostimulatorydomain. Thus, the expression product of said heterologous nucleic acidmolecule may be targeted specifically to predetermined compartments suchas periplasma, outer membrane, etc. The heterologous nucleic acidmolecule may code for a fusion protein.

According to one embodiment the heterologous nucleic acid molecule isinserted into the SPI2 locus, preferred, into an sse gene and, morepreferred, into sseC, sseD and/or sseE, in particular, sseC.

The insertion may be a polar insertion or an unpolar insertion.Generally, the introduction of an unpolar insertion is preferred, sinceit allows for the expression of the remaining genes of a polycistronicgene cluster, which can be used for the generation of carriers havingdifferent grades of attenuation.

Attenuated live Salmonella vaccines are used as carriers for specificantigens from heterologous pathogens, e.g. Helicobacter, etc., thusacting as a multivalent vaccine. The heterologous antigens are providedby a gene expression cassette (GEC) that is inserted by geneticengineering into the genome of an attenuated Salmonella strain.Preferentially, insertion of the gene expression cassette is targeted toone of the indicated virulence genes, thereby causing an insertionalmutation as described in previous paragraph. In another applicationform, expression of the heterologous genes in the gene expressioncassette is regulated by trans-acting factors encoded by atrans-activator cassette (TC) or an invertase cassette performing a2-phase variable expression mode. Preferentially, the insertion of thetrans-activator cassette is targeted to a second chosen virulence gene,which is then inactivated. Alternatively, the gene expression cassetteor the trans-activator cassette or the invertase cassette can beintroduced into the Salmonella genome by transposon-mediated insertion,which has no attenuation effect.

The principles of genetic engineering are required to generate eitherdeletion or insertional mutations in Salmonella virulence genes.Generally, a suicide plasmid carrying a mutated virulence gene cassettecontaining a selective marker cassette (SMC) either alone or incombination with a gene expression cassette or a trans-activatorcassette or the invertase cassette is introduced into the receptorSalmonella strain by conjugation. The original virulence gene isreplaced with the mutated virulence gene cassette via homologousrecombination, and the suicide plasmid, unable to replicate in theSalmonella receptor strain, becomes rapidly depleted. Successfullyrecombined Salmonella can be selected based on properties (such as, butnot limited to, antibiotic resistance) conferred by the product of thegene(s) within the selective marker cassette. The mutated virulence genecassette comprises DNA sequences that are homologous to the genome ofthe receptor Salmonella strain where the original virulence gene islocalised. In the case where the original virulence gene is to becompletely deleted, only those genomic DNA sequences that border theoriginal virulence gene (indicated as flanking regions) are included inthe mutated virulence gene cassette. The general architecture of amutated virulence gene cassette includes at each end a DNA sequence ofat least 50 nucleotides, ideally 200-250 nucleotides, that is homologousto the genome segment where the original virulence gene is localised.These DNA sequences flank a selective marker cassette and the othercassettes, such as the gene expression cassette (GEC) or thetrans-activator cassette (TC) or the invertase cassette. As indicatedabove, these cassettes are used to generate insertional mutations whichdisrupt original gene expression. For in-frame deletions, a selectivemarker cassette is preferentially used.

The selective marker cassette (SMC) principally consists of a genemediating resistance to an antibioticum which is able to inactivate thereceptor Salmonella strain but which is actually not used in thetreatment of Salmonellosis. Alternatively, another selectable marker canbe used. The selective marker cassette is inserted in-frame in thetargeted virulence gene and, consequently, the expression of the markergene is under the control of the virulence gene promoter. Alternatively,the cassette is inserted within a polycistronic transcriptional unit, inwhich case the marker gene is under control of the promoter for thisunit. In another application, the selective marker gene is under controlof its own promoter; in this case a transcriptional terminator isincluded downstream of the gene. The selective marker is needed toindicate the successful insertion of the mutated virulence gene cassetteinto the genome of the receptor Salmonella strain. Furthermore, theantibiotic resistance marker is needed to facilitate the pre-clinicalimmunological assessment of the various attenuated Salmonella strains.In another application form, the selective marker is flanked by directrepeats, which, in the absence of selective pressure, lead to therecombinatorial excision of the selective marker cassette from thegenome, leaving the short sequence of the direct repeat. Alternatively,the selective marker cassette can be completely removed by recombinantDNA technology. Firstly, the selective marker cassette is removed byadequate restriction endonuclease from the original mutated virulencegene cassette on the suicide plasmid leaving the flanking regionsequences which are homologous to the Salmonella genome. The suicideplasmid is then transfered into the attenuated receptor Salmonellastrain by conjugation where the SMC-depleted mutated virulence genecassette replaces the SMC-carrying mutated virulence gene cassette byrecombination. After removal of the selective marker, the attenuatedSalmonella strain is free for the application in humans. Transcriptionalterminator sequences are generally included in the cassettes when polarmutations are established.

The gene expression cassette (GEC) comprises elements that allow,facilitate or improve the expression of a gene. In a functional mode thegene expression cassette additionally comprises one or more geneexpression units derived from either complete genes from a heterologoussource or fragments thereof, with a minimal size of an epitope. Multiplegene expression units are preferentially organised as a concatemericstructure. The genes or gene fragments are further geneticallyengineered, such that the resulting proteins or fusion proteins areexpressed in the cytosol, in the periplasm, surface displayed orsecreted. Furthermore the genes or gene fragments can be fused with DNAsequences encoding immunologically reactive protein portions, e.g.cytokines or attenuated bacterial toxins. The genes or gene fragmentsare either controlled in a one-phase mode from a promoter within thegene expression cassette or in a 2-phase mode or indirectly by atrans-activator cassette (TC). In the one-phase mode the promoter ispreferentially a Salmonella promoter that is activated, i.e. induced, byenvironmental signals but also constitutive promoters of differentstrength can be used. In the 2-phase mode, the expression of the genecassette is controlled by an invertase that derived from an invertasecassette. The invertase catalyses the inversion of a DNA segmentcomprising the gene cassette. The DNA segment is flanked on each end byan inverted repeat which is the specific substrate for the invertasefinally causing two orientation of the gene cassette with respect to thegene expression cassette promoter. In the ON-orientation the genecassette is correctly placed allowing transcription of the genecassette. In OFF, the orientation of the gene cassette is incorrect andno transcription occurs. The invertase cassette comprises of aninvertase that is controlled by a constitutive promoter or a Salmonellapromoter induced or derepressed by environmental signals.

Heterologous antigens encoded within the gene expression cassette can beexpressed under the control of a promoter, e.g. a tissue-specificpromoter, which may be constitutive or inducible. The expression can beactivated in a target cell, whereby a signal is transmitted from thetarget cell to the interior of the Salmonella cell, which signal inducesthe expression. The target cell, for example, can be a macrophage. Theexpression product may comprise a targeting domain or immunostimulatorydomain, e.g. in the form of a fusion protein. The heterologous proteinitself also may be a fusion protein. The heterologous antigens can beoptionally expressed as cytosolic, periplasmic, surface displayed orsecretory proteins or fusion proteins in order to achieve an efficaciousimmune response. The antigen encoding sequences may be fused toaccessory sequences that direct the proteins to the periplasm or outermembrane of the Salmonella cell or into the extracellular milieu. If theheterologous polypeptides are secreted, secretion can occur using a typeIII secretion system. Secretion by the SPI2 type III secretion system issuitable. Proteins that are destined for the cytosolic compartment ofthe Salmonella do not need accessory sequences, in this case, naturallyoccurring accessory sequences must be removed from the genes encodingsuch antigens.

The accessory sequences for the periplasmatic compartment of Salmonellacomprise a DNA sequence deduced from the amino-terminally localisedsignal peptide of a heterologous protein naturally translocated via thegeneral secretion pathway, e.g. CtxA, etc.

The accessory sequences for the outer membrane compartment of Salmonellapreferentially comprise DNA sequences deduced from the functionallyrelevant portions of a type IV secretory (autotransporter) protein, e.g.AIDA or IgA protease. The appropriate fusion protein contains anamino-terminally localised signal peptide and, at the carboxy-terminus,a β-barrel shaped trans-membrane domain to which the foreign passengerprotein is coupled via a spacer that anchors the passenger protein tothe bacterial surface.

The accessory sequences for secretion into the extracellular milieucomprise DNA sequences deduced from proteins naturally secreted by thetype III secretion system. In a generally functional fusion protein, theheterologous antigen is fused in the centre of a protein naturallysecreted by the type III pathway or at the carboxy-terminal end of therespective protein.

The transactivator cassettes (TC) provide activators which generallyimprove expression of the heterologous antigens encoded by the variousgene expression cassettes. Such activators either directly (RNApolymerase) or indirectly (transcriptional activator) act on thetranscription level in a highly specific order. Preferentially, theexpression of such activators are controlled by Salmonella promoterswhich are induced in vivo by environmental signals. In anotherapplication form the synthesis of the activator within thetransactivator cassette is regulated in a 2-phase mode. The invertaseexpressed by the invertase cassette places the activator encoding DNAfragment in two orientations with respect to the transcriptionalpromoter. In the ON-orientation the activator gene is in the correcttranscriptional order. In the OFF-modus the activator is incorrectlyorientated and no expression occurs.

In the simple system, the gene product of the transactivator cassetteexerts its effect directly on the promoter present in the geneexpression cassette, directly activating or de-repressing expression ofthe heterologous gene. In the complex system, activation of the promoterin the heterologous gene expression cassette is dependent upon two ormore interacting factors, at least one of which (encoded in thetransactivator cassette) may be regulated by external signals. Furthercomplexity is found in cascade systems, in which the external signaldoes not directly exert its effect on the transactivator cassette, butrather through a multi-step process, or in which the gene product of thetransactivator cassette does not directly exert its effect on theheterologous gene expression cassette, but rather through a multi-stepprocess.

According to still another aspect, the present invention is anattenuated gram-negative cell comprising the SPI2 gene locus,characterized by a lack of at least one SPI2 polypeptide, wherein saidlack results in an attenuation/reduction of virulence compared to thewild type of said cell. Preferably, said missing SPI2 polypeptide is oneor more effector polypeptide, secretion apparatus polypeptide, chaperonpolypeptide or regulatory polypeptide. Furthermore, said attenuated cellmay be a carrier which then is characterized by the presence of at leastone heterologous peptide or polypeptide having immunogenic properties.

A further aspect of the present invention is a pharmaceuticalcomposition which comprises as an active agent an immunologicallyprotective living vaccine which is an attenuated gram-negative cell orcarrier according to the invention. The pharmaceutical composition willcomprise additives such as pharmaceutically acceptable diluents,carriers and/or adjuvants. These additives are known to the personskilled in the art. Usually, the composition will administered to apatient via a mucosa surface or via or via the parenteral route.

Further aspects of the present invention include a method for thepreparation of a living vaccine, which comprises providing a livinggram-negative cell comprising the SPI2 locus and inactivating at leastone gene of the SPI2 locus to obtain an attenuated gram-negative cell ofthe invention, and optionally inserting at least one heterologousnucleic acid molecule coding for an antigen to obtain a carrieraccording to the invention. A further aspect pertains to a method forthe preparation of a living vaccine composition comprising formulatingan attenuated cell or a carrier according to the invention in apharmaceutically effective amount together with pharmaceuticallyacceptable diluents, carriers and/or adjuvants. A further aspect of theinvention relates to a method for the detection of an attenuated cell ora carrier according to the invention, comprising providing a samplecontaining said cell and detecting a specific property not present in awild type cell. Methods for detecting a specific property of theattenuated cell or carrier, which is not present in wild type, are knownto the person skilled in the art. For example, if this specific propertyof the attenuated cell comprises a deletion of one or more parts of theSPI2 locus, then the presence of said cell can be detected by providinga pair of specific primers which are complementary to sequences flankingthis deletion and amplifying a fragment of specific length usingamplification methods such as PCR. Methods for detecting the presence ofan inventive carrier comprise PCR amplification of an inserted fragmentor a fragment spanning the insertion boundary, hybridization methods orthe detection of the heterologous expression product or of a selectivemarker.

A further aspect of the invention is a method for establishing a libraryof attenuated gram-negative cells or carriers, respectively, accordingto the invention. The method comprises the preparation of attenuatedrecombinant vaccine strains, each having a different mutation in theSPI2 locus which results in a different degree of attenuation. Thepathogenicity or virulence potential of said strains can then bedetermined using known methods such as determination of the LD50, andthe strains are rated according to the different pathogenicities, i.e. adifferent grade of attenuation. Preferably, the method comprises alsothe determination of other parameters of interest such as theimmunogenicity or the immuno-stimulatory response raised in a host.Methods for determining the immuno-stimulatory potential are known tothe person skilled in the art and some of them are described in Example6. Preferably, the immuno-stimulatory potential of the inventiveattenuated cells or carriers is determined at humoral, cellular and/ormucosal level. In this way it is possible to establish a library ofattenuated cells or carriers having a predetermined attenuation degreeand predetermined immuno-stimulatory properties. Thus, for eachapplication, the strain having the desired properties can be selectedspecifically. For example, it will be usually preferred to select astrong attenuated strain for administration to patients which receiveimmunosuppressive drugs.

In a similar way, the invention allows for the establishment oflibraries of attenuated carriers having defined pathogenicities andoptionally immunogenicities. The establishment of a carrier libraryadditionally will comprise the determination of the antigen presentationof said carrier strains to a host, whereby a panel of different carriersstrains will be obtained having defined properties with respect topathogenicity, immuno-stimulatory potential of carrier antigens andimmuno-stimulatory potential of the heterologous antigen.

Another aspect of the invention is the use of the attenuated cell orcarrier according to the invention for the preparation of a drug for thepreventive or therapeutic treatment of an acute or chronic diseasecaused essentially by a bacterium or virus. For example, for theprevention or treatment of a Salmonella infection one will administer anattenuated Salmonella cell to raise the immune response of an affectedpatient. Similarly, a carrier according to the invention may be used forthe preparation of a drug for the preventive or therapeutic treatment ofa tumor.

The individual immuno-protective potential of each of the establishedrecombinant Salmonella vaccine strains is determined in a mouse modelusing a pathogenic Salmonella typhimurium as the challenge strain.

-   -   Determination of the virulence potential of the recombinant        Salmonella vaccine strain: (1) Competitive index or LD50; (2)        Systemic prevalence in blood, liver and spleen strictly        excluded.    -   Determination of the immuno-stimulatory potential of the carrier        strain with a cytosolically expressed heterologous test        antigen: (1) Single oral immunisation and subsequent evaluation        of the short- and long-term immune response: (a) analysis of the        humoral immune response profile, (b) analysis of the mucosal        immune response profile, (c) analysis of the cellular immune        response profile; (2) Multiple oral immunisations and subsequent        evaluation of the short- and long-term immune response: (a)        analysis of the humoral immune response profile, (b) analysis of        the mucosal immune response profile, (c) analysis of the        cellular immune response profile.    -   Determination of the immuno-stimulatory potential of the carrier        strain for the delivery of heterologous DNA (DNA vaccination).

Preferentially, the Salmonella acceptor strain has a broad host range,exhibiting significant pathogenicity in both animals and humans.Ideally, this is a Salmonella strain that is strongly pathogenic formice, such as S. typhimurium. After successful development of therecombinant Salmonella vaccine strain, the strain is directly applicablefor use in both animals and humans. If such an ideal Salmonella acceptorstrain is not satisfactory for the respective host, other host-specificSalmonella must be selected, such as S. typhi for humans.

Other aspects of the invention relate to the use of a nucleic acidmolecule as shown in FIG. 21A or B or one of the FIGS. 22A-Q, optionallymodified as described hereinabove or of a vector as describedhereinabove for the preparation of an attenuated cell, a living vaccineor a carrier for the presentation of an antigen to a host and to the useof the Salmonella SPI2 locus for the preparation of an attenuated cell,a living vaccine or preferably a carrier for the presentation of anantigen to a host. In this context the term “Salmonella SPI2 locus”refers to any nucleic acid sequence, coding or not coding, and to theexpression product of coding sequences.

A still further aspect of the present invention is the use of avirulence gene locus of a gram-negative cell for the preparation of acarrier for the presentation of an antigen to a host.

Another aspect of the invention relates to a method of therapeuticallyor prophylactically vaccinating an animal, e.g. a mammal, e.g. a human,against a chronic disease caused primarily by a infectious organismincluding preparation and administering a vaccine of the invention.

Still another aspect of the present invention is an isolated nucleicacid molecule comprising a nucleic acid of at least 100 nucleotides a)of the nucleic acid sequence of one of FIGS. 24A, B, b) of a nucleicacid sequence which under stringent conditions hybridizes with thenucleic acid sequence of one of FIGS. 24A, B.

In particular, said aspect relates to said nucleic acid molecule whichis capable of inducing the expression of a nucleic acid sequence condingfor a peptide or polypeptide operatively linked to said nucleic acidmolecule.

The in vivo inducible promoter Pivi comprises a DNA fragment whichcarries sequences for an operator and a transcriptional promoter. Suchin vivo inducible promoter can be identified by applying an adequatereporter gene approach. Two of such in vivo inducible promoters havebeen identified within the SPI2 locus which initiate expression of thessaBCDE operon (promoter A2) and the sseABsscAsseCDEsscBsseFG operon(promoter B), respectively. These promoters are induced by a regulativesystem comprising the ssrA and ssrB gene products. This regulativesystem is part of the SPI2 locus responsible for the activation ofadditional SPI2 locus genes. The regulative system is activated inmacrophages by environmental signal(s) via sensor protein SsrA. The SsrBprotein finally binds at a defined DNA sequence which initiatestranscription through the RNA polymerase.

In an application form the DNA fragment comprising operator/promotersequences is inserted in front of an invertase gene or an activator geneor a gene expression cassette, thereby executing an in vivo inducibleexpression in bacteria carrying at least the ssrA and ssrB genes or thecomplete SPI2 locus.

Thus, in a further aspect, the invention relates to an expression systemfor the in vivo inducible expression of a heterologous nucleic acid in atarget cell, comprising a carrier cell for said heterologous nucleicacid, wherein said carrier cell comprises (a) a polypeptide having theamino acid sequence shown in FIG. 23P (ssrA) or a functional homologuethereof, (b) a polypeptide having the amino acid sequence shown in FIG.23Q (ssrB) or a functional homologue thereof, and (c) the nucleic acidmolecule of one of FIGS. 24A, B or a functional homologue thereof, asdescribed above.

The target cell may be any suitable cell but preferably it is amacrophage. The carrier cell preferably is a Salmonella cell. The targetcell may also comprise one or more of the elements described above suchas selective marker cassettes, gene expression cassettes, transactivatorcassettes, invertase cassettes and/or insertion cassettes. Furthermore,it may comprise a heterologous nucleic acid, in particular, theheterologous nucleic acids may be inserted into a gene expressioncassette, thus rendering the GEC functional.

A still further aspect of the invention relates to the use of a nucleicacid molecule comprising at least 100 nucleotides of the nucleic acidsequence shown in one of FIGS. 24A, B or hybridizing therewith andhaving promoter activity, for the in vivo inducible expression of aheterologous nucleic acid molecule.

A further aspect of the present invention is the use of said nucleicacid molecule for the detection of in vivo inducible promoters.

EXPERIMENTAL PROCEDURES

The strains, material, and methods used in the type III secretion systemof the Salmonella Pathogenicity Island 2 (SPI2) work described above areas follows:

Mice

Female BALB/c (H-2^(d)) of 6-12 weeks of age were maintained understandard conditions according to institutional guidelines. This studywas approved by an ethic committee for animal use in experimentalresearch.

Bacterial Strains, Phages and Plasmids

The bacterial strains, phages and plasmids used in this study are listedin Table 1. Unless otherwise indicated, bacteria were grown at 37° C. inLuria Bertani (LB) broth or agar, supplemented with ampicillin (50μg/ml), kanamycin (50 μg/ml), or chloramphenicol (50 μg/ml) whereappropriate. Eukaryotic cells were grown in RPMI 1640 supplemented with10% of foetal calf serum (FCS), 100 U/ml penicillin, 50 μg/mlstreptomycin, 5×10⁻⁵ M 2-mercaptoethanol and 1 mM L-glutamine (GIBCOBRL; Prisley, Scotland). To achieve constitutive expression of β-gal,the plasmid pAH97 (Holtel et al., 1992) was electroporated into thecarrier strains as described elsewhere (O'Callaghan and Charbit, 1990).

TABLE 1 Phages, plasmids and bacterial strains used in this work. Phage,plasmid or strain Description Reference Phages λ1 clone from a libraryof S. typhimurium Shea et al., genomic DNA in λ1059 1996 λ2 clone from alibrary of S. typhimurium Shea et al., genomic DNA in λ1059 1996 λ5clone from a library of S. typhimurium Shea et al., genomic DNA in λ10591996 Plasmids pBluescriptKS+, Amp^(r); high copy number cloningStratagene, pBluescriptSK+ vectors Heidelberg pUC18 Amp^(r); high copynumber cloning vector Gibco-BRL, Eggenstein pT7-Blue Amp^(r); high copynumber cloning vector Novagen, Heidelberg pCVD442 suicide vectorDonnenberg et al., 1991 pACYC184 Cm^(r), Tet^(r); low copy numbercloning Chang and vector Cohen, 1978 pGPL01 R6K ori, Amp^(r);λpir-dependent Gunn and suicide vector for luc fusions Miller, 1996pLB02 R6K ori, Amp^(r); λpir-dependent Gunn and suicide vector for lucfusions Miller, 1996 pGP704 R6K ori, Amp^(r); λpir-dependent Miller andsuicide vector Mekalanos, 1988 pKAS32 Amp^(r); λpir-dependent suicidevector; Skorupski rpsl^(s) and Taylor, 1996 pNQ705 R6K ori, Cm^(r);λpir-dependent suicide Forsberg et vector al., 1994 pSB315 Kan^(r),Amp^(r) Galán et al., 1992 p1-6 Amp^(r), 4.8 kb PstI/BamHI fragment ofthis work λ1 in pT7-Blue p1-20 1.7 kb BamHI/HincII fragment of p1-6 thiswork in pKS+ p1-21 aphT cassette in EcoRV site of p1-20 this work p1-22XbaI/KpnI insert of p1-21 in pKAS32 this work p2-2 Amp^(r), 5.7 kb BamHIfragment of λ2 in this work pUC18 p2-20 1.6 kb HindIII/HincII fragmentof p2-2 this work in HindIII/SmaI-digested pKS+ p2-21 aphT cassette inHincII site of p2-20 this work p2-22 insert of p2-21 in pKAS32 this workp2-50 3.7 kb BamHI/KpnI fragment of p2-2 in this work pKS+ p5-2 Amp^(r);5.7 kb EcoRI fragment of λ5 in this work pKS+ p5-30 3.0 kb PstI/EcoRIfragment of p5-2 in this work pUC18 p5-31 aphT cassette in EcoRV site ofp5-30 this work p5-33 SphI/EcoRI insert of p5-31 in pGP704 this workp5-4 Amp^(r); 5.8 kb HindIII fragment of λ5 in this work pSK+ p5-40 4.5kb SstI/HindIII fragment of p5-2 in this work pKS+ p5-41 aphT cassettein SmaI site of p5-40 this work p5-43 KpnI/SstI insert of p5-41 inpNQ705 this work p5-5 Amp^(r); PstI digestion of p5-4 and this workreligation of the larger fragment p5-50 2.6 kb BamHI/ClaI fragment ofp5-2 in this work pKS+ p5-51 aphT cassette in HindIII site of p5-50 thiswork after Klenow fill-in p5-53 XbaI/SalI insert of p5-51 in pGP704 thiswork p5-60 ClaI-digestion of p5-2 and religation of this work largerfragment p5-8 Amp^(r), 2.2 kb PstI/HindIII fragment of this work p5-2 inpSK+ psseA Cm^(r); sseA in pACYC184 this study psseB Cm^(r); sseB inpACYC184 this study psseC Cm^(r); sseC in pACYC184 this work E. colistrains DH5α see reference Gibco-BRL S17-1 λpir λpir phage lysogen (seereference) Miller and Mekalanos, 1988 CC118 λpir λpir phage lysogen (seereference) Herrero et al., 1990 XL1-Blue see reference Stratagene S.typhimurium strains NCTC12023 wild-type Colindale, UK CS015phoP-102::Tn10d-Cm Miller et al., 1989 CS022 phoP^(c) Miller et al.,1989 P2D6 ssaV::mTn5 Shea et al., 1996 P3F4 ssrA::mTn5 Shea et al., 1996P4H2 hilA::mTn5 Monack et al., 1996 P6E11 spaRS::mTn5 Shea et al., 1996P8G12 ssrB::mTn5 Shea et al., 1996 P9B6 ssaV::mTn5 Shea et al., 1996P9B7 ssaT::mTn5 Shea et al., 1996 P11D10 ssaJ::mTn5 Shea et al., 1996NPssaV ssaV::aphT, Km^(r); non-polar mutation Deiwick et al., 1998 HH100sseAΔ:::aphT, Km^(r); non-polar mutation this study HH101 HH100containing psseA this study HH102 sseBΔ:::aphT, Km^(r); non-polarmutation this study HH103 HH102 containing psseB this study HH107sseFΔ:::aphT, Km^(r); non-polar mutation this study HH108 sseG::aphT,Km^(r); non-polar mutation this study MvP102 ΔsseEsscB,_Km^(r);non-polar mutation this work MvP103 sseC::aphT, Km^(r); non-polarmutation this work MvP103[psseC] MvP103 containing psseC this workMvP131 ssaB::luc in S. typhimurium this work NCTC12023 MvP127 sseA::lucin S. typhimurium this work NCTC12023 MvP239 sipC::lacZY, EE638 in S.typhimurium Hueck et al., NCTC12023 1995; this work MvP244 ssaB::luc inS. typhimurium P8G12 this work MvP266 ssaH::luc in S. typhimurium thiswork NCTC12023 MvP284 ssrA::aphT, Km^(r); non-polar mutation this workMvP320 ssrB::aphT, Km^(r); non-polar mutation this work MvP337 in-framedeletion in sseC this work MvP338 in-frame deletion in sseD this workMvP339 in-frame deletion in sscB this work MvP340 in-frame deletion inssrA this work SL7207 S. typhimurium 2337-65 hisG46, gift from DEL407aroA::TnI(Tc-s) B. A. D. Stocker III-57 sseC Δ sseC this work

Example 1 Distribution of the Pathogenicity Island SPI-2 withinDifferent Salmonella Strains

The presence of open reading frames of the SPI-2 region in variousSalmonella isolates and E. coli K-12 was analyzed by Southernhybridization as shown in Table 2.

TABLE 2 Prevalence of SPI-2 genes in various Salmonella ssp. deducedfrom representative gene probes serovar/ Species subspec. serotype ssrABORF S. enterica I typhimurium + + S. enterica I typhi + + S. entericaII + + S. enterica IIIa + + S. enterica IIIb + + S. enterica IV + + S.enterica VI + + S. enterica VII + + S. bongori 66:z41:— − + S. bongori44:z48:— − + E. coli K-12 − −

Presence or absence of hybridizing bands is indicated by + or −,respectively.

Hybridization

Genomic DNA of various Salmonella strains and E. coli K-12 was preparedas previously described (Hensel et al., 1997a). For Southernhybridization analysis, genomic DNA was digested with EcoRI or EcoRV,fractionated on 0.6% agarose gels and transferred to Hybond N⁺ membranes(Amersham, Braunschweig). Various probes corresponding to the SPI-2region were obtained as restriction fragments of the subcloned insert ofλ1. Probes corresponding to ORF 242 and ORF 319 were generated by PCRusing primer sets D89 (5′-TTTTTACGTGAAGCGGGGTG-3′) (SEQ ID NO:44) andD90 (5′-GGCATTAGCGGATGTCTGACTG-3′) (SEQ ID NO:45), and D91(5′-CACCAGGAACCATTTTCTCTGG-3′) (SEQ ID NO:46) and D92(5′-CAGCGATGACGATATTCGACAAG-3′) (SEQ ID NO:47), respectively. PCR wasperformed according to the specifications of the manufacturer(Perkin-Elmer, Weiterstadt). PCR products were submitted to agarose gelelectrophoresis and fragments of the expected size were recovered andpurified. Hybridization probes were labeled using the DIG labelingsystem as described by the manufacturer (Boehringer, Mannheim).

Example 2 Characterization of sse Genes and Construction of sseC::aphT,sseD::aphT and sseEΔ Mutant S. Typhimurium Strains MvP103, MvP101 andMvP102

Organization of sse and ssc Genes

In order to characterize SPI2 genetically and functionally, a centralregion of the pathogenicity island (FIG. 1A) has been cloned andsequenced. DNA fragments covering the region between ssaC and ssaJ weresubcloned in plasmids p5-2 and p5-4 as indicated in FIG. 1C. Thearrangement and designation of genes in the 8 kb region between ssaC andssaK is shown in FIG. 1B. This sequence will be available from the EMBLdatabase under accession number AJ224892 in the near future. Thesequenced region extends the open reading frame (ORF) of a gene encodinga putative subunit of the type III secretion apparatus referred to asspiB (Ochman et al., 1996). For consistency with the universalnomenclature for type III secretion system subunits (Bogdanove et al.,1996) and the nomenclature of other SPI2 genes (Hensel et al., 1997b),this gene has been designated ssaD. The deduced amino acid sequence ofssaD is 24% identical to YscD of Y. enterocolitica. This is followed byan ORF with coding capacity for a 9.3 kDa protein, 34% identical to YscEof Y. enterocolitica. Therefore, this gene is designated ssaE. Asequence of 263 bp separates ssaE and a set of nine genes, several ofwhich encode proteins with sequence similarity to secreted effectorproteins or their chaperones from other pathogens. These genes areseparated by short intergenic regions or have overlapping reading framesand it is likely that some are co-transcribed and translationallycoupled. Therefore, the genes with similarity to those encodingchaperones were designated sscA and sscB, and the others sseA-E. Theamino acid sequence deduced from sscA shows 26% identity/49% similarityover 158 amino acid residues to SycD, the product of IcrH of Y.pseudotuberculosis which acts as a secretion-specific chaperone for YopBand YopD (Wattiau et al., 1994). The amino acid sequence deduced fromsscB shows 23% identity/36% similarity over 98 amino acid residues toIppi of Shigella flexneri. IppI is a chaperone for S. flexneri invasionproteins (Ipas) (Baundry et al., 1988). As is the case for the secretionchaperones SycD, Ippl and SicA (Kaniga et al., 1995), SscB has an acidicpI (Table 3), whereas SscA has an unusually high pI of 8.8. SseB is 25%identical/47% similar to EspA of EPEC over the entire length of the 192amino acid residue protein (FIG. 2 b). SseD is 27% identical/51% similarto EspB of EPEC over 166 amino acid residues. SseC has sequencesimilarity to a class of effector proteins involved in the translocationof other effectors into the target host cell. These include YopB of Y.enterocolitica, EspD of EPEC and PepB of Pseudomonas aerugunosa. SseC isapproximately 24% identical/48% similar to both EspD of EPEC and YopB ofY. enterocolitica (FIG. 2 a). EspD and YopB have two hydrophobic domainsthat are predicted to insert into target cell membranes (Pallen et al.,1997). SseC contains three hydrophobic regions that could representmembrane-spanning domains. Other features of these predicted effectorproteins are shown in Table 1. Using the TMpredict program (Hofmann andStoffel, 1993), transmembrane helices are predicted for all the effectorproteins apart from SseA which is very hydrophilic. Alignments of SseCto homologs in other pathogens are shown in FIG. 2 b. Conserved aminoacids are mainly clustered in the central, more hydrophobic portion ofthe protein, but unlike YopB, there is no significant similarity to theRTX family of toxins. The conserved residues in SseD are present mainlyin the N-terminal half of the protein. Comparison of the deduced aminoacid sequences of sseABCDEF with entries in the PROSITE database did notreveal the presence of any characteristic protein motifs. We subjectedthe predicted amino acid sequences of the sse genes to searches usingthe programs COIL and MULTICOIL as described by Pallen et al. (1997).SseA and SseD are predicted to have one trimeric coil each, and SseC ispredicted to have two trimeric coils (Table 3). Since EspB and EspD arepredicted to have one and two trimeric coils, respectively (Pallen etal., 1997), this provides further evidence that these proteins arefunctionally related.

TABLE 3 Features of predicted proteins. Protein M_(r) (kDa) pI Tmpredictions Predicted coils SseA 12.5 9.3 hydrophilic at least one(trimer) SseB 21.5 4.7 one transmembrane helix none SseC 52.8 6.3 threetransmembrane at least two (trimers) helices SseD 20.6 4.8 threetransmembrane at least one (trimer) helices SseE 16.3 9.7 onetransmembrane helix none SscA 18.1 8.8 hydrophilic none SscB 16.4 4.7hydrophilic noneExpression of SPI2 GenesGeneration of Antibodies Against Recombinant SPI2 Proteins

In order to monitor the expression of the SPI2 genes sseB, sscA andssaP, a Western blot analysis of total bacterial cells with polyclonalantibodies raised against recombinant SPI2 proteins SseB, SscA, and SsaPwas performed.

Protein gel electrophoresis and Western blotting were performed asdescribed elsewhere (Laemmli, 1970 and Sambrook et al., 1989). Plasmidsfor the expression of recombinant SPI2 protein were constructed bycloning the individual SPI2 genes in plasmids pQE30, pQE31 or pQE32(Qiagen, Hilden) in order to generate in-frame fusion to the N-terminal6His tag. Recombinant SPI2 genes were expressed in E. coli M 5 [pREP](Qiagen) and purified by metal chelating chromotography according torecommendations of the manufacturer (Qiagen). For immunisation, about 1mg of recombinant SPI2 proteins were emulsified with complete andincomplete Freund's adjuvant for primary and booster immunizations,respectively. Rabbits were immunized subcutaneously according tostandard protocols (Harlow and Lane, 1988). SPI2 proteins were detectedwith antisera raised against recombinant SPI2 proteins afterelectrophoretical separation of proteins from total cells and transferedonto a nitrocellulose membrane (Schleicher and Schuell) using a‘Semi-Dry’ blotting device (Bio-Rad) according to the manufacturersmanual. Bound antibody was visualized using a secondaryantibody-alkaline phosphatase conjugate according to standard protocols(Harlow and Lane).

Generation of Reporter Gene Fusions:

Fusions of the reporter gene firefly luciferase (luc) to various genesin SPI2 were obtained using the suicide vectors pLB02 and pGPL01 (Gunnand Miller, 1996), which were kindly provided by Drs. Gunn and Miller(Seattle).

For the generation of a fusion to ssaB, a 831 bp EcoRV fragment of p2-2was subcloned in EcoRV digested pSK⁺. For the generation of atranscriptional fusion to sseA, a 1060 bp SmaI/HincII fragment of p5-4was subcloned in pSK⁺. The inserts of the resulting constructs wererecovered as a EcoRI/KpnI fragment and ligated with EcoRI/KpnI digestedreporter vectors pGPL01 and pLB02. For the generation of atranscriptional fusion to ssaJ, a 3 kb SmaI/KpnI fragment of p5-2 wasdirectly subcloned in pGPL01 and pLB02.

Constructs with transcriptional fusions of SPI2 genes to luc were thanintegrated into the chromosome of S. typhimurium by mating between E.coli S17-1 λpir harbouring the respective construct and a spontaneousmutant of S. typhimurium resistant to 100 μg×ml⁻¹ nalidixic acid andselection for exconjugants resistant to carbenicillin and nalidixicacid. The targeted integration in SPI2 (for constructs using pGLP01) orthe zch region (for constructs using pLB02) was confirmed bySouthernanalysis. Fusions were then moved into a mouse-passaged strainof S. typhimurium NCTC12023 by P22 transduction according to standardprocedures (Maloy et al., 1996).

Assay of Reporter Genes

β-galactosidase activities of reporter gene fusions were determinedaccording to standard procedures (Miller, 1992).

Bacterial strains harbouring firefly luciferase fusions to SPI2 genes(strain MvP127, sseA::luc, strain MvP131, ssaB::luc, strain MvP266,ssaH::luc) were grown in medium with various Mg²⁺ concentrations. Theluciferase activity of aliquots of the cultures was determined using thePromega (Heidelberg) luciferase assay kit or custom made reagentsaccordingly. Briefly, bacteria were pelleted by centrifugation for 5min. at 20000×g at 4° C. and resuspended in lysis buffer (100 mM KHPO₄,pH 7.8, 2 mM EDTA, 1% Triton X-100, 5 mg×ml⁻¹ bovine serum albumin, 1 mMDTT, 5 mg×ml⁻¹ lysozyme). Lysates were incubated for 15 min at roomtemperature with repeated agitation and subjected to a freeze/thawcycle. Aliquots of the lysates (25 μl) were transferred to microtiterplates (MicroFLUOR, Dynatech) and immediately assayed after addition of50 μl luciferase reagent (20 mM Tricine-HCl, pH 7.8, 1.07 mM(MgCO₃)₄Mg(OH)₂, 100 μM EDTA, 33.3 mM DTT, 270 μM_Li₃-coenzyme A, 470 μMD(−)-luciferin, 530 μM Mg-ATP) for photon emission using the TriLuxMicroBeta luminometer (Wallac, Turku). All assays were done intriplicates and repeated on independent occasions.

Expression of SPI2 Genes such as ssaB and ssaH is Induced by low Mg²⁺Concentrations of the Growth Medium

S. typhimurium wild-type strain and strains harbouring luc reporter-genefusions to ssaB (strain MvP131) and to ssaH (strain MvP266) were grownto mid-log phase (OD at 600 nm of about 0.5) in minimal media containinghigh amounts of Mg²⁺ (10 mM MgCl₂). This medium is referred to as mediumG. Bacteria were recovered by centrifugation, washed three times inminimal medium containing 8 μM Mg²⁺. This medium is referred to asmedium F. Bacteria were resuspended in medium F or medium G and growthat 37° C. was continued. Aliquots of the cultures of strains MvP131 andMvP266 were withdrawn at the several different time points indicated andsubjected to analysis of luciferase activity. Aliquots of the wild-typestrain were withdrawn at the same time points. Protein from totalbacterial cells was separated by SDS-PAGE and transferred tonicrocellulose membranes. These blots were incubated with antibodiesraised against recombinant SsaP and SscA protein in order to detectproteins synthesized after the magnesium concentration shift in themagnesium concentration. After shifting bacteria from a growth mediumwith high amounts of Mg²⁺ to a medium with limiting amounts of Mg²⁺, theexpression of SPI2 genes was highly induced. This induction can bemonitored by using the reporter gene luc fused into different positionsof SPI2. Furthermore, proteins synthesized after induction of SPI2 weredetected by Western Blots. However, even in the presence of high amountsof Mg²⁺, a low level of expression of SPI2 genes was observed.

Expression of SPI2 Genes Such as sseA and ssaB is Modulated by PhoP/PhoQRegulation

No expression of sseb or sscA was observed during growth in various richmedia, or cell culture media with or without serum. However, low amountof SsaP were detected after growth in Lysogeny Broth (LB) or other richmedia such as brain heart infusion (BHI). Growth in minimal mediumcontaining less than 30 μM Mg²⁺ induces the expression of SPI2 genes.Such effect of the Mg²⁺ concentration has so far only been observed forPhoP/PhoQ-regulated genes. This observation is in contrast to a previousreport by Valdivia and Falkow (1997) who postulated that SPI2 geneexpression is independent of PhoP/PhoQ. However, in a PhoP^(c)(constitutive) strain background (CSO22, Miller et al., 1989) expressionof SPI2 genes was not constitutive but still dependent on the Mg²⁺concentration of the medium. This indicates that SPI2 gene expression ismodulated by PhoP/PhoQ, but that further regulatory elements such asSsrA/B are needed.

DNA Cloning and Sequencing

DNA preparations and genetic manipulations were carried out according tostandard protocols (Sambrook et al, 1989). Plasmid DNA transformation ofbacterial cells was performed by electroporation (O'Callaghan andCharbit, 1990).

Clones harbouring fragments of SPI2 were identified from a library ofgenomic DNA of S. typhimurium in λ 1059 which has been describedpreviously (Shea et al., 1996). The sse and ssc genes were subclonedfrom clone λ5 on a 5.7 kb EcoRI fragment (p5-2) and a 5.8 kb HindIIIfragment (p5-4) in pBluescriptKS + as indicated in FIG. 1 and Table 1.

DNA sequencing was performed using a primer-walking strategy. Thedideoxy method (Sanger et al., 1977) was applied using the Pharmacia T7sequencing system for manual sequencing and the dye terminator chemistryfor automatic analysis on a ABI377 sequencing instrument. Assembly ofcontigs from DNA sequences was performed by means of AssemblyLign andMacVector software (Oxford Molecular, Oxford). For further sequenceanalyses, programs of the GCG package version 8 (Devereux et al., 1984)were used on the HGMP network.

Construction of Non-polar Mutations

The construction of non-polar mutations in sseC (MvP103), sseD (MvP101)and sseE (MvP102) are described below. All chromosomal modificationswere confirmed by PCR and Southern hybridization analysis (Southern,1975, J. Mol. Biol. 98: 503-517).

-   -   Mutant MvP103, sseC. A 2.6 kb fragment was recovered after BamHI        and ClaI digestion of p5-2 and subcloned in BamHI/ClaI-digested        pBluescript II KS +. The resulting construct termed p5-50 was        digested by HindIII, blunt ended using the Klenow fragment of        DNA polymerase and ligated to the aphT cassette. A 900 bp HincII        fragment of pSB315 containing an aminoglycoside        3′-phosphotransferase gene (aphT) from which the transcriptional        terminator had been removed (Galán et al., 1992) was ligated in        the same orientation into the blunted-ended HindIII site of        plasmid p5-50. After transformation of E. coli XL-1 Blue and        selection for resistance against kanamycin and carbenicillin (50        μg/ml each) one clone has been chosen and the harbouring plasmid        isolated. This plasmid was termed p5-51 and its identity        confirmed by restriction analysis. It was further digested with        SalI and XbaI and the insert of 3.5 kb was ligated to        SalI/XbaI-digested pGP704. This plasmid was electroporated        into E. coli CC118 λpir and the transformants selected for        resistance to kanamycin and carbenicillin (50 ig/ml each). As        done before, one clone was chosen, its plasmid with the        according DNA fragment in pGP704, termed p5-53, isolated and        confirmed by restriction analysis. Plasmid p5-53 was        electroporated into E. coli S 17-1 λpir and transferred into S.        typhimurium NCTC12023 (resistant to nalidixic acid, 100 μg/ml)        by conjugation as has been described previously (de Lorenzo and        Timmis, 1994). Exconjugants in which the sseC gene had been        replaced by the cloned gene disrupted by insertion of the aphT        cassette were selected by resistance to kanamycin and nalidixic        acid (100 μg/ml). The resulting exconjugants were finally tested        for a lactose-negative phenotype and their sensitivity to        carbenicillin. Selected clones were further examined by        Southernblot analysis. In order to exclude possible mutations        which have been acquired during the cloning procedure the        mutated sseC allele was transferred into a fresh Salmonella        background by P22 transduction (described by Maloy et al.,        1996). The resulting Salmonella strain MvP103 was examined for        the presence of the resistance cassette within the sseC gene by        the use of PCR. Amplification was performed by using the primers        E25 (5′-GAAATCCCGCAGAAATG-3′) (SEO ID NO:48) and E28        (5′-AAGGCGATAATATAAAC-3′) (SEO ID NO:49). The resulting fragment        had a size of 1.6 kb for S. typhimurium wild-type and 2.5 kb for        strain MvP103.

For complementation of non-polar mutations in sseC, the correspondinggenes were amplified by PCR from genomic DNA using a series of primerscorresponding to the region 5′ of the putative start codons and to the3′ ends of the genes. These primers introduced BamHI restriction sitesat the termini of the amplified genes. After digestion with BamHI, thegenes were ligated to BamHI-digested pACYC184 (Chang and Cohen, 1978)and transferred into E. coli DH5α. The orientation of the insert wasdetermined by PCR, and in addition, DNA sequencing was performed toconfirm the orientation and the correct DNA sequence of the inserts.Plasmids with inserts in the same transcriptional orientation as theTetr gene of pACYC184 were selected for complementation studies andelectroporated into the S. typhimurium strains harbouring correspondingnon-polar mutations.

-   -   Mutant MvP101, sseD. A 3.0 kb fragment was recovered after PstI        and EcoRI digestion of p5-2 and subcloned in PstI/EcoRI-digested        pUC18 The resulting construct termed p5-30 was digested by EcoRV        and treated with alkaline phosphatase. The aphT cassette was        isolated as described above and ligated to the linearized        plasmid p5-30 in the same orientation in the unique EcoRV site.        After transformation of E. coli XL-1 Blue and selection against        kanamycin and carbenicillin (50 μg/ml each) one clone has been        chosen and the harbouring plasmid isolated. This plasmid was        termed p5-31 and its identity confirmed by restriction analysis.        p5-31 was further digested with SphI and EcoRI, a 4.0 kb        fragment isolated and ligated to SphI/EcoRI-digested pGP704.        This plasmid was electroporated into E. coli CC118 λpir and        transformants selected to kanamycin and carbenicillin (50 μg/ml        each). As done before, one clone was chosen, its plasmid with        the according DNA fragment in pGP704, termed p5-33, isolated and        confirmed by restriction analysis. Plasmid p5-33 was        electroporated into E. coli S17-1 λpir and transferred into S.        typhimurium NCTC12023 (resistant to nalidixic acid) by        conjugation as has been described previously (de Lorenzo and        Timmis, 1994). Exconjugants in which the sseD gene had been        replaced by the cloned gene disrupted by insertion of the aphT        cassette were selected by resistance to kanamycin and nalidixic        acid (100 μg/ml). The resulting exconjugants were finally tested        for a lactose-negative phenotype and their sensitivity to        carbenicillin. Selected clones were further examined by        Southernblot analysis. In order to exclude possible mutations        which might have been accumulated during the cloning procedure        the mutated sseD allele was transferred into a fresh Salmonella        background by P22 transduction (described by Maloy et al.,        1996). The resulting Salmonella strain MvP1O1 was examined for        the presence of the resistance cassette within the sseD gene by        the use of PCR. Amplification was performed by using the primers        E6 (5′-AGAGATGTAflAGATAC-3′) (SEQ ID NO:50) and E28        (5′-AAGGCGATAATATAAAC-3′) (SEQ ID NO:49). The resulting fragment        had a size of 0.8 kb for S. typhimurium wild-type was used and        1.7 kb in the case of strain MvP101.    -   Mutant MvP102, deletion or parts of sseE and sscB. A 4.5 kb        fragment was recovered after SstI and HindIII digestion of p5-2        and subcloned in SstI/HindIII-digested pKS +. The resulting        construct termed p5-40 was digested by SmaI, digested with        alkaline phospatase and ligated to the aphT cassette in the same        orientation into the unique SmaI site created in the sseE/sseB        deletion plasmid p5-40 as described above. After transformation        of E. coli XL-1 Blue and selection against kanamycin and        carbenicillin (50 μg/ml each) one clone was chosen and the        harbouring plasmid isolated. This plasmid was termed p5-41 and        its identity confirmed via restriction analysis. It was further        digested with KpnI and SstI and the insert was ligated to        KpnI/SstI-digested pNQ705. This plasmid was electroporated        into E. coli CC118 λpir and transformed bacteria selected to        kanamycin and chloramphenicol (50 μg/ml each). As done before,        one clone was chosen, its plasmid with the according DNA        fragment in pNQ705, termed p5-43, isolated and confirmed by        restriction analysis. The resulting plasmid was used to transfer        the mutated gene onto the Salmonella chromosome as described        above. Resulting clones have been further examined by        Southernblot analysis. To exclude possible mutations which might        have been acquired during the cloning procedure the mutated        sseE/sscB allele was transferred into a fresh Salmonella        background by P22 transduction (described by Maloy et al.,        1996). The resulting Salmonella strain MvP102 was examined for        the presence of the resistance cassette within the sseE/sseB        gene by the use of PCR. Amplification was performed by using the        primers E6 (5′-AGAGATGTATTAGATAC-3′) (SEQ ID NO:50) and E4        (5′-GCAATAAGAGTATCAAC-3′) (SEQ ID NO:51). The resulting fragment        had a size of 1.6 kb for S. typhimurium wild-type and a size of        1.9 kb for strain MvP102.        Construction of Mutant Strains Carrying In-frame Deletions in        sseC, sseD and sscB:

Based on the observation that a non-polar in sseE did not result in asignificant attenuation of virulence in the mouse model (Hensel et al.,1998), the generation of a deletion mutant for the sseE gene is not ofinterest for the generation of carrier strains.

Construction of an In-frame Deletion in sseC, Mutant MvP337

A deletion of 158 bp between codon 264 and 422 of sseC was generated.Plasmid p5-2 was digested by ClaI and the larger fragment containing thevector portion was recovered and self-ligated to generate p5-60. Plasmidp5-60 was linearized by digestion with HindIII, which cuts once withinthe sseC gene. Primers sseC-del-1 (5′-GCT AAG CTT CGG CTC AAA TTG TTTGGA AAA C-3′) (SEQ ID NO:52) and sseE-del-2 (5′-GCT AAG CTT AGA GAT GTATTA GAT ACC-3′) (SEQ ID NO:53) were designed to introduce HindIII sites.PCR was performed using linearized p5-60 as template DNA. The TAQPLUS®polymerase (Pyrococcus furiosus DNA polymerase + recombinant T.Aquaticus YT1 polymerase) (Stratagene) was used according to theinstructions of the manufacturer. Reactions of 100 μl volume were set upusing 10 μl of 10× TAQPLUS® Precision buffer containing magnesiumchloride, 0.8 μl of 100 mM dNTPs, 250 ng DNA template (linearized p5-8),250 ng of each primer and 5 U of TAQPLUS® DNA polymerase (Pyrococcusfuriosus DNA polymerase + recombinant T. Aquaticus YT1 polymerase). PCRwas carried out for 35 cycles of: 95° C. for 1 minute, 60° C. for 1minute, 72° C. for 6 minutes. Then a final step of 72° C. for 10 minuteswas added. 10 μl of the PCR reaction were analyzed. A product of theexpected size was recovered, digested by HindIII, self-ligated, and theligation mixture was used to transform E. coli DH5α to resistance tocarbenicillin. Plasmids were isolated from transformants and theintegrity of the insert and the deletion was analyzed by restrictiondigestion and DNA sequencing. The insert of a confirmed construct wasisolated after digestion with XbaI and KpnI and ligated toXbaI/KpnI-digested vector pKAS32. The resulting construct was used totransform E. coli S17-1 λpir to resistance to carbenicillin, andconjugational transfer of the plasmid to S. typhimurium (NaI^(R),Strep^(R)) was performed according to standard procedures (de Lorenzoand Timmis, 1994). Exconjugants that had integrated the suicide plasmidby homologous recombination were selected by resistance to nalidixicacid and carbenicillin, and screened for sensitivity to streptomycin.Such clones were grown in LB to OD600 of about 0.5 and aliquots wereplated on LB containing 250 μg/ml streptomycin to select for colonieswhich had lost the integrated plasmid and undergone allelic exchange.Clones resistant to streptomycin but sensitive to carbenicillin wereused for further analysis. Screening of mutants with a deletion withinthe sseC locus was performed by PCR using primers sseC-For (5′-ATT GGATCC GCA AGC GTC CAG AA-3′) (SEQ ID NO:54) and sseC-Rev (5-TAT GGA TCCTCA GAT TAA GCG CG-3′) (SEQ ID NO:55). Amplification of DNA from clonescontaining the wild-type sseC allele resulted in a PCR product of 1520bp, use of DNA from clones harbouring a sseC allele with an internaldeletion resulted in a PCR product of 1050 bp. The integrity of clonesharbouring the sseC deletion was further confirmed by Southern analysisof the sseC locus. Finally, the sseC locus containing the internalin-frame deletion was moved into a fresh strain background of S.typhimurium by P22 transduction (Maloy et al., 1996) and the resultingstrain was designated MvP 337.

Construction of an In-frame Deletion in sseD, Mutant Strain MvP338

A deletion of 116 bp between codon 26 and 142 of sseD was generated.Plasmid p5-2 was digested by HindIII/PstI and a fragment of 2.1 kb wasisolated and subcloned in HindIII/PstI-digested vector pBluescript SK +.The resulting construct was designated p5-8. p5-8 was linearized bydigestion with EcoRV, which cuts twice within the sseD gene. PrimerssseD-del-1 (5′-ATA GAA TTC GGA GGG AGA TGG AGT GGA AG -3′) (SEQ IDNO:56) and sseD-del-2 (5′-ATA GAA TTC GAA GAT AAA GCG ATT GCC GAC-3′)(SEQ ID NO:57) were designed to introduce EcoRI sites. PCR was performedusing linearized p5-8 as template DNA. The TAQPLUS® polymerase(Pyrococcus furiosus DNA polymerase + recombinant T. Aquaticus YT1polymerase) (Stratagene) was used according to the instructions of themanufacturer. Reactions of 100 μl volume were set up using 10 μl of 10×TAQPLUS® Precision buffer containing magnesium chloride, 0.8 μl of 100mM dNTPs, 250 ng DNA template (linearized p5-8), 250 ng of each primerand 5 U of TAQPLUS® DNA polymerase (Pyrococcus furiosus DNA polymerase +recombinant T. Aquaticus YT1 polymerase). PCR was carried out for 35cycles of: 95° C. for 1 minute, 60° C. for 1 minute, 72° C. for 5minutes. Then a final step of 72° C. for 10 minutes was added. 10 μl ofthe PCR reaction were analyzed. A product of the expected size wasrecovered, digested by EcoRI, self-ligated, and the ligation mixture wasused to transform E. coli DH5á to resistance to carbenicillin. Plasmidswere isolated from transformants and the integrity of the insert and thedeletion was analyzed by restriction mapping and DNA sequencing. Theinsert of a confirmed construct was isolated after digestion with XbaIand KpnI and ligated to XbaI/KpnI-digested vector pKAS32. The resultingconstruct was used to transform E. coli S17-1 λpir to resistance tocarbenicillin, and conjugational transfer of the plasmid to S.typhimurium (Nal^(R), Strep^(R)) was performed according to standardprocedures (de Lorenzo and Timmis, 1994). Exconjugants that hadintegrated the suicide plasmid by homologous recombination were selectedby resistance to nalidixic acid and carbenicillin, and screened forsensitivity to streptomycin. Such clones were grown in LB to OD600 ofabout 0.5 and aliquots were plated on LB containing 250 ig/mlstreptomycin to select for colonies which had lost the integratedplasmid and undergone allelic exchange. Clones resistant to streptomycinbut sensitive to carbenicillin were used for further analysis. Screeningof mutants with a deletion within the sseD locus was performed by PCRusing primers sseD-For (5′-GAA GGA TCC ACT CCA TCT CCC TC-3′) (SEQ IDNO:58) and sseD-Rev (5-GAA GGA TCC ATT TGC TCT ATT TCT TGC-3′) (SEQ IDNO:59). Amplification of DNA from clones containing the wild-type sseDallele resulted in a PCR product of 560 bp, use of DNA from clonesharbouring a sseD allele with an internal deletion resulted in a PCRproduct of 220 bp. The integrity of clones harbouring the sseD deletionwas further confirmed by Southernanalysis of the sseD locus. Finally,the sseD locus containing the internal in-frame deletion was moved intoa fresh strain background of S. typhimurium by P22 transduction (Maloyet al., 1996) and the resulting strain was designated MvP338.

Construction of an In-frame Deletion in sscB, Mutant Strain MvP339

A deletion of 128 bp between codon 32 and 160 of sscB was generated. A 3kb BglII fragment of plasmid p5-2 was ligated into the BamHI site ofpBluescript KS + to generate plasmid p5-70. Plasmid p5-70 was linearizedby digestion with NcoI, which cuts once within the sscB gene. PrimerssscB-del-1 (5′-ATG GGA TCC GAG ATT CGC CAG AAT GCG CAA-3′) (SEQ IDNO:60) and sscB-del-2 (5′-ATG GGA TCC ACT GGC ATA AAC GGT TTC CGG-3′)(SEQ ID NO:61) were designed to introduce BamHI sites. PCR was performedusing linearized p5-70 as template DNA. The TAQPLUS® polymerase(Pyrococcus furiosus DNA polymerase+recombinant T. Aquaticus YT1polymerase) (Stratagene) was used according to the instructions of themanufacturer. Reactions of 100 μl/volume were set up using 10 μl of 10×TAQPLUS® Precision buffer containing magnesium chloride, 0.8 μl of 100mM dNTPs, 250 ng DNA template (linearized p5-70), 250 ng of each primerand 5 U of TAQPLUS® DNA polymerase (Pyrococcus furiosus DNApolymerase+recombinant T. Aquaticus YT1 polymerase). PCR was carried outfor 35 cycles of: 95° C. for 1 minute, 60° C. for 1 minute, 72° C. for 6minutes. Then a final step of 72° C. for 10 minutes was added. 10 μl ofthe PCR reaction were analyzed. A product of the expected size wasrecovered, digested by BamHI, self-ligated, and the ligation mixture wasused to transform E. coli DH5á to resistance to carbenicillin. Plasmidswere isolated from transformants and the integrity of the insert and thedeletion was analyzed by restriction analysis and DNA sequencing. Theinsert of a confirmed construct was isolated after digestion with XbaIand KpnI and ligated to XbaI/KpnI-digested vector pKAS32. The resultingconstruct was used to transform E. coli S17-1 λpir to resistance tocarbenicillin, and conjugational transfer of the plasmid to S.typhimurium (Nal^(R), Strep^(R)) was performed according to standardprocedures (de Lorenzo and Timmis, 1994). Exconjugants that hadintegrated the suicide plasmid by homologous recombination were selectedby resistance to nalidixic acid and carbenicillin, and screened forsensitivity to streptomycin. Such clones were grown in LB to OD600 ofabout 0.5 and aliquots were plated on LB containing 250 ig/mlstreptomycin to select for colonies which had lost the integratedplasmid and undergone allelic exchange. Clones resistant to streptomycinbut sensitive to carbenicillin were used for further analysis. Screeningof mutants with a deletion within the sseC locus was performed by PCRusing primers sscB-For (5′-ATT GGA TCC TGA CGT AAA TCA TTA TCA-3′) (SEQID NO:62) and sscB-Rev (5-ATT GGA TCC TTA AGC AAT AAG TGA ATC-3′) (SEQID NO:63). Amplification of DNA from clones containing the wild-typesscB allele resulted in a PCR product of 480 bp, use of DNA from clonesharbouring a sscB allele with an internal deletion resulted in a PCRproduct of 100 bp. The integrity of clones harbouring the sseC deletionwas further confirmed by Southernanalysis of the sscB locus. Finally,the sscB locus containing the internal in-frame deletion was moved intoa fresh strain background of S. typhimurium by P22 transduction (Maloyet al., 1996) and the resulting strain was designated MvP339.

Construction of a Deletion Mutation in the sseC Gene

In a further approach the complete sequence of the chromosomal sseC genewas deleted by allelic replacement with a deleted copy of the gene. Thedeletion was constructed in a suicide plasmid (pCVD442 (Donnenberg etal., 1991). First, two DNA fragments flanking the sseC gene (fragment A,carrying artificial SalI and XbaI sites at its 5′ and 3′ ends,respectively; and fragment B, carrying artificial XbaI and SacI sites atits 5′ and 3′ ends, respectively) were amplified by PCR. Theoligonucleotides used for PCR were: 1.) sseDelfor1GCTGTCGACTTGTAGTGAGTGAGCAAG (SEQ ID NO:70) (3′ nucleotide corresponds tobp 941 in included sequence: FIG. 21A); 2.) sseCDelrev2GGATCTAGATTTTAGCTCCTGTCAGAAAG (SEQ ID NO:71) (3′ nucleotide correspondsto bp 2585 in included sequence, oligo binds to reverse strand); 3.)sseCDelfor2 GGATCTAGATCTGAGGATAAAAATATGG (SEQ ID NO:72) (3′ nucleotidecorresponds to bp 4078 in included sequence); 4.) sseDelrev1GCTGAGCTCTGCCGCTGACGGAATATG (SEQ ID NO:73) (3′ nucleotide corresponds tobp 5592 in included sequence, oligo binds to reverse strand). Theresulting PCR fragments were fused together via the XbaI site. Theresulting fragment was cut with SalI and SacI and cloned into pCVD442cut with SalI and SacI. The resulting plasmid was introduced into S.typhimurium NCTC12023 by conjugation and chromosomal integrants of theplasmid into the sseC locus were selected for by the plasmid-encodedampicillin resistance marker. In a second step, clones which had lostthe plasmid were screened for by loss of ampicillin resistance. Theresulting clones were tested for chromosomal deletion of the sseC geneby PCR, and deletion of a 1455 bp fragment, comprising the entire sseCopen reading frame, was confirmed. This ΔsseC mutant strain was namedIII-57ΔsseC.

Construction of a sseC-aroA Double Mutant

In order to construct a double mutant which can serve as a prototype fora live attenuated vaccine, the sseC:aphT(Km′) marker from MvP103 wastransferred by P22 phage transduction into S. typhimurium SL7207 (hisG46DEL407 [aroA544:Tn10], Tc^(R)) a strain carrying a stable deletion inthe aroA gene.

Example 3 Invasion and Intracellular Growth in Tissue Culture

Intramacrophage Replication of Mutant Strains

Several strains which are defective in their ability to replicate insidemacrophages and macrophage-like cell lines have been tested, asmacrophage survival and replication are thought to represent animportant aspect of Salmonella pathogenesis in vivo (Fields et al.,1986). It has been reported previously that a number of SPI2 mutantstrains were not defective for survival or replication within RAWmacrophages (Hensel et al., 1997b) but subsequent experiments haverevealed that some SPI2 mutants can be shown to have a replicationdefect if aerated stationary phase bacterial cultures opsonized withnormal mouse serum are used (see also accompanying paper: Cirillo etal., 1998). The increase in cfu for different strains in RAW macrophagesover a 16 h period is shown in FIG. 3. Replication defects were observedfor strains carrying mutations in ssaV (encoding a component of thesecretion apparatus), sseB and sseC and to a lesser extent for strainscarrying mutations in sseE. Partial complementation of this defect wasachieved with strains harbouring plasmids carrying functional copies ofsseb and sseC, HH103 and MvP103[psseC], respectively. The ability ofSPI2 mutant strains to replicate inside the J774.1 macrophage cell line(FIG. 4A) and in periodate-elicited peritoneal macrophages from C3H/HeNmice (FIG. 4B) has also been tested. Similar replication defects of S.typhimurium carrying transposon or non-polar mutations in SPI2 geneswere observed, regardless of the phagocyte cell-type examined, althoughthe peritoneal elicited cells had superior antimicrobial activitycompared to either cell line.

Macrophage Survival Assays

RAW 264.7 cells (ECACC 91062702), a murine macrophage-like cell line,were grown in Dulbecco's modified Eagle's medium (DMEM) containing10%_(foetal) calf serum (FCS) and 2 mM glutamine at 37° C. in 5% CO₂ .S. typhimurium strains were grown in LB to stationary phase and dilutedto an OD₆₀₀ of 0.1 and opsonized for 20 min in DMEM containing 10%normal mouse serum. Bacteria were then centrifuged onto macrophagesseeded in 24 well tissue culture plates at a multiplicity of infectionof approximately 1:10 and incubated for 30 min. Following infection, themacrophages were washed twice with PBS to remove extracellular bacteriaand incubated for 90 min (2h post-infection) or 16 h in mediumcontaining gentamicin (12 μg/ml). Infected macrophages were washed twicewith PBS and lysed with 1% Triton X-100 for 10 min and appropriatealiquots and dilutions were plated onto LB agar to enumerate cfu.

Survival of opsonized S. typhimurium strains in J774.1 cells (Ralph etal., 1975) or C3H/HeN murine peritoneal exudate cells (from CharlesRiver Laboratories, Wilmington, Mass.) was determined essentially asdescribed by DeGroote et al. (1997), but without the addition ofinterferon-γ. Briefly, peritoneal cells harvested in PBS withheat-inactivated 10%_(foetal) calf serum 4 days after intraperitonealinjection of 5 mM sodium periodate (Sigma, St. Louis, Mo.) were platedin 96-well flat-bottomed microtiter plates (Becton-Dickinson, FranklinLakes, N.J.) and allowed to adhere for 2 h. Non-adherent cells wereflushed out with prewarmed medium containing 10% heat-inactivated_(foetal) calf serum. In previous studies, we have established that >95%of the cells remaining after this procedure are macrophages. S.typhimurium from aerated overnight cultures was opsonized with normalmouse serum and centrifuged onto adherent cells at an effector to targetratio of 1:10. The bacteria were allowed to internalize for 15 min, andwashed with medium containing 6 μg/ml gentamicin to kill extracellularbacteria. At 0 h and 20 h, cells were lysed with PBS containing 0.5%deoxycholate (Sigma, St. Louis, Mo.), with plating of serial dilutionsto enumerate colony-forming units.

Example 4 Evaluation of Safety in the S. typhimurium Mouse Model ofSalmonellosis

Virulence Tests with Strains Carrying Non-polar Mutations

DNA sequence analysis suggested that the sse genes might encode effectorproteins of the secretion system, but apart from a possible polar effectfrom a transposon insertion in sscA no strains carrying mutations inthese genes were recovered in the original STM screen for S. typhimuriumvirulence genes using mTn5 mutagenesis (Hensel et al., 1995), and theirrole in virulence was unclear. To address this question, strainscarrying non-polar mutations in sseC, sseD and sseEsscB (FIG. 1) havebeen constructed and subjected to virulence tests. Table 4 shows thatall mice inoculated with strains carrying mutations sseC and sseDsurvived a dose of 1×10⁴ cfu, three orders of magnitude greater than theLD₅₀ of the wild-type strain, which is less than 10 cfu when theinoculum is administered by the i.p. route (Buchmeier et al., 1993; Sheaet al, 1996). The same strains containing a plasmid carrying thecorresponding wild-type allele were also inoculated into mice at a doseof 1×10⁴ cfu. No mice survived these infections, which shows that eachmutation can be complemented by the presence of a functional copy ofeach gene, and that each of these genes plays an important role inSalmonella virulence. Strains carrying non-polar mutations in sseEsscBcaused lethal infections when approximately 1×10⁴ cells of each strainwere inoculated into mice by the i.p. route (Table 4) and were analyzedin more detail by a competition assay with the wild-type strain in mixedinfections (five mice/test) to determine if they were attenuated invirulence. The competitive index, defined as the output ratio of mutantto wild-type bacteria, divided by the input ratio of mutant to wild-typebacteria, shows that the sseEsscB mutant was not significantly differentto that of a fully virulent strain carrying an antibiotic resistancemarker, which implies that this gene does not play a significant role insystemic Salmonella infection of the mouse.

TABLE 4 Virulence of S. typhimurium strains in mice. Mouse Mousesurvival survival after after in- inoculation^(a) oculation^(a) withwith mutant + bacterial complementing Competitive Strain Genotype strainplasmid index in vivo NCTC120 wild-type 0/5 n.d. 0.98^(b) 23 MvP101ΔsseD::aphT 5/5 n.d. >0.01 MvP102 ΔsseEsscB::aphT 4/4 n.d. 0.79 MvP103sseC::aphT 5/5 0/5 >0.01 (oral) >0.01 (i.p.) ^(a)Mice were inoculatedintraperitoneally with 1 × 10⁴ cells of each strain ^(b)Result ofcompetition between wild-type strain NCTC12023 and a virulent mTn5mutant identified in the STM screen.

Example 5 Vaccination with the sseC::aphT, and AsseD::aphT mutant S.Typhimurium Strains MvP103 and MvP101

Strains Carrying Non-polar Mutations as Live Vaccine Carriers

To confirm the suitability of the MvP101 and MvP103 mutants as livevaccine carriers their level of attenuation was evaluated by determiningthe LD₅₀ after oral inoculation in mice. Groups of 10 mice were fed withserial dilutions of either MvP110, MvP103 or the wild-type parentalstrain NCTCNCTC12023 and dead animals were recorded within a period of10 days postinfection. The obtained results demonstrated that bothmutants are highly attenuated when given orally to BALB/c mice (LD₅₀above 10⁹) when compared with the parental strain (LD₅₀=6.9×10⁵ CFU).After intraperitoneal inoculuation the LD₅₀ of S. typhimurium NCTC12023wild-type in BALB/c is 6 bacteria, and the LD₅₀ of MvP103 in BALB/c is2.77×10⁶ after intraperitoneal inoculation. The mutation can becomplemented by psseC, but no LD₅₀ determination for the complementedmutant strain was performed. LD₅₀ of MvP101 in BALB/c is 3.54×10⁶ afterintraperitoneal inoculation. A partial complementation by plasmid p5-K1was possible. An intraperitoneal LD₅₀ for MvP101 [p5-K1] of 8.45×10² wasdetermined. (Description of p5-K1: a 3.2 kb PstI fragment of p5-2containing sseC′sseDsseEsscBsseF′ was subcloned in low copy numbercloning vector pWSK29).

Determination of the LD₅₀

Doses ranging from 10⁵ to 10⁹ CFU of either S. typhimurium NCTCNCTC12023(wild-type) or the mutants MvP103 and MvP101 were orally inoculated intogroups of 10 mice and survival was recorded over 10 days.

LD₅₀ of S typhimurium wild-type and mutant strains MvP101 and MvP103after intraperitoneal infection was determined by inoculation of dosesranging from 10¹ to 10⁷ CFU into groups of 5 female BALB/c mice of 6-8weeks of age. Survival was recorded over a period of three weeks. TheLD₅₀ dose of the challenge strains was calculated by the method of Reedand Muench (Reed and Muench, 1938).

Immunization Protocols

For vaccination, bacteria were grown overnight until they reach mediumlog phase. Then, they were harvested by centrifugation (3,000×g) andresuspended in 5% sodium bicarbonate. Mice were immunized four times at15 day intervals by gently feeding them with the bacterial suspension(10⁹ CFU/mouse) in a volume of approximately 30 μl. Control mice werevaccinated with the carrier, lacking plasmid.

Cytotoxicity Assay

Spleen cells were obtained from mice 14 days after the last immunizationand 2×10⁶ effector cells were restimulated in vitro for 5 days incomplete medium supplemented with 20 U/ml of rIL-2 and 20 μM of the βGP1peptide (β-gal p876-884, TPHPARIGL), which encompasses theimmunodominant H-2L^(d)-restricted β-gal epitope. After restimulation,the assay was performed using the [³H]-thymidine incorporation method.In brief, 2×10⁶ of P815 cells per ml were labelled with [³H]-thymidinefor 4 h in either complete medium or complete medium supplemented with20 μM of βGP1 peptide and used as target cells. Following washing, 2×10⁵labelled targets were incubated with serial dilutions of effector cellsin 200 μl of complete medium for 4 h at 37° C. Cells were harvested andspecific lysis was determined as follows: [(retained c.p.m. in theabsence of effectors)−(experimentally retained c.p.m. in the presence ofeffectors)/retained c.p.m. in the absence of effectors]×100.

Example 6 Evaluation of the Induced Immune Response

Induction of Mucosal Immune Responses After Oral Vaccination

To achieve protection against mucosal pathogens using live Salmonellacarriers, elicitation of an efficient mucosal response is highlydesirable. Therefore, the presence of β-gal-specific antibodies inintestinal washes from mice immunized with either MvP101, MvP103 orSL7207 carrying pAH97 was investigated 52 days after immunization. Asshown in FIG. 5., immunization with all three carriers stimulate theproduction of significant amounts of β-gal-specific IgA and, to a lesserextent, favor the transudation of antigen-specific IgG in the intestinallumen. No statistically significant differences were observed among themucosal responses to the different recombinant clones.

Cellular Immune Responses Triggered After Oral Immunization with sseCand sseD Mutants Expressing β-gal

To evaluate the efficacy of the antigen-specific T cell responsesgenerated in immunized mice, spleen cells were enriched in CD4 + T cellsand restimulated in vitro during four days with β-gal. As shown in FIG.6, although antigen-specific CD4 +-enriched spleen cells were generatedafter vaccination with the three carriers, MvP103 and MvP101 weresignificantly more efficient than SL7207 (P 0.05) at triggering specificcellular immune response. In contrast, cells isolated from miceimmunized with the carrier alone failed to proliferate in the presenceof β-gal.

To investigate the Th-type of immune response triggered by immunization,the content of IFN-γ, IL-2, IL-4, IL-5, IL-6 and IL-10 was measured inthe supernatant fluids of restimulated cells. The results demonstratedthat a predominant Th1 response pattern was induced in mice immunizedwith all the carriers. IFN-γ was the only cytokine with significantlyincreased levels in comparison to those observed in supernatants fromspleen cells isolated from mice immunized with plasmidless carriers(FIG. 7). Interestingly, in agreement with the IgG isotype patterns, thelevels of IFN-γ detected in supernatants from cells of mice immunizedwith MvP103 [pAH97] were significantly higher (P 0.05) than those fromanimals receiving either MvP101 [pAH97] or SL7207 [pAH97] (FIG. 7).

Antigen-specific antibody responses generated in mice orally immunizedwith the attenuated S. typhimurium vaccine carriers expressing the modelantigen β-gal

Groups of mice were immunized with the recombinant strains MvP101[pAH97] and MvP103 [pAH97]. To estimate the efficacy of the prototypesanother group was vaccinated with the well-established carrier strainSL7207 [pAH97]. The abilities of the different carriers to induce asystemic humoral response was determined by measuring the titer ofβ-gal-specific antibodies in the serum of vaccinated mice. As shown inFIG. 8, significant titers of β-gal-specific IgG and IgM antibodies weredetected at day 30 in all vaccinated animals. In contrast to the IgMtiters which reach a plateau at day 30, the titers of IgG steadilyincreased until day 52 from immunization when the experiment wasconcluded. Although all tested carriers exhibit an excellentperformance, the MvP103 mutant was the most efficient at inducinganti-β-gal IgG antibodies (P 0.05). No significant levels ofβ-gal-specific IgA were detected in mice immunized with any of the threerecombinant clones (data not shown).

To determine the subclass distribution of the anti-β-gal IgG, serumsamples were analyzed for specific levels of IgG1, IgG2a, IgG2b andIgG3. The results shown in FIG. 9 demonstrate that the mainβ-gal-specific IgG isotype present in sera of all immunized mice wasIgG2, suggesting of a predominant Th1 response. Interestingly, a lowerconcentration of IgG1 (P 0.05) was observed in mice immunized withMvP103 than in those receiving MvP101 and SL7202, indicating a similarresponse pattern in animals immunized with the last two carriers.

Sample Collection

Serum samples were collected at different time points and monitored forthe presence of β-gal-specific antibodies. At day 52 after immunization,intestinal ravages were obtained by flushing the small intestine with 2ml of PBS supplemented with 50 mM EDTA, 0.1% bovine serum albumin and0.1 mg/ml of soybean trypsin inhibitor (Sigma). Then, the ravages werecentrifuged (10 min at 600×g) to remove debris, supernatants wereremoved and supplemented with phenylmethylsulfonyl fluoride (10 mM) andNaN₃, and stored at −20° C.

Antibody Assays

Antibody titres were determined by an enzyme-linked immunosorbent assay(ELISA). Briefly, 96 well Nunc-Immuno MaxiSorp™ assay plates (Nunc,Roskilde, Denmark) were coated with 50 μl/well β-gal (5 μg/ml) incoating buffer (0.1 M Na₂HPO₄, pH 9.0). After overnight incubation at 4°C., plates were blocked with 10% FCS in PBS for 1 h at 37° C. Serialtwo-fold dilutions of serum in FCS-PBS were added (100 μl/well) andplates were incubated for 2 h at 37° C. After four washes with PBS-0.05%Tween 20, secondary antibodies were added: biotinylated γ-chain specificgoat anti-mouse IgG, μ-chain specific goat anti-mouse IgM, α-chainspecific goat anti-mouse IgA antibodies (Sigma, St. Louis, Mo.) or, todetermine IgG subclass, biotin-conjugated rat anti-mouse IgG1, IgG2a,IgG2b and IgG3 (Pharmingen) and plates were further incubated for 2 h at37° C. After four washes, 100 μl of peroxidase-conjugated streptavidin(Pharmingen, St. Diego, Calif.) were added to each well and plates wereincubated at room temperature for 1 h. After four washes, reactions weredeveloped using ABTS [2,2′-azino-bis-(3-ethybenzthiazoline-6-sulfonicacid)] in 0.1 M citrate-phosphate buffer (pH 4.35) containing 0.01%H₂O₂. Endpoint titers were expressed as the reciprocal log₂ of the lastdilution which gave an optical density at 405 nm 0.1 unit above thevalues of the negative controls after a 30 min incubation.

To determine the concentration of total Ig present in the intestinalravages, serial dilutions of the corresponding samples were incubated inmicrotiter plates that had been coated with goat anti-mouse IgG, IgM andIgA as capture antibodies (100 μg/well, Sigma) and serial dilutions ofpurified mouse IgG, IgM and IgA (Sigma) were used to generate standardcurves. Detection of antigen-specific Ig was performed as describedabove.

Induction of Antigen-specific CTL Responses in Mice Orally Immunizedwith the Carrier Strains Expressing β-gal

The elicitation of MHC class I restricted responses are particularlyimportant for protection against many intracellular pathogens andtumors. It has been shown that antigen-specific CD8 + CTL can begenerated both in vitro and in vivo after immunization with recombinantSalmonella spp. expressing heterologous antigens. Therefore, weconsidered it important to determine whether the tested carriers werealso able to trigger a β-gal-specific CTL response. Spleen cells werecollected from mice vaccinated with either MvP101 [pAH97], MvP103[pAH97] or SL7207 [pAH97] at day 52 from immunization and restimulatedin vitro with βGP1-pulsed syngenic spleen cells for 5 days. As shown inFIG. 10, the spleen cells from mice immunized with either of the threeconstructs induced significant lysis of βGP1-loaded target cellscompared with unloaded controls. The more efficient responses wereobserved using the carrier strain MvP103. The lysis was mediated byCD8 + T cells since the cytotoxic activity was completely abrogated whenCD8 + T effector cells were depleted (data not shown).

Cytokine Determination

Culture supernatants were collected from proliferating cells on days 2and 4, and stored at −70° C. The determination of IL-2, IL-4, IL-5,IL-6, IL-10 and IFN-γ was performed by specific ELISA. In brief, 96-wellmicrotiter plates were coated overnight at 4° C. with purified ratanti-mouse IL-2 mAb (clone JESG-1A12), anti-IL-4 mAb (clone 11B11),anti-IL-5 mAb (clone TRFK5), anti-IL-6 mAb (clone MP5-20F3), anti-IL-10mAb (clone JES5-2A5), and anti-IFN-γ mAb (clone R4-6A2) (Pharmingen).After three washes, plates were blocked and two-fold dilutions ofsupernatant fluids were added. A standard curve was generated for eachcytokine using recombinant murine IL-2 (rIL-2), rIL-4, rIL-5, rIL-6,rIFN-γ, and rIL-10 (Pharmingen). Plates were further incubated at 4° C.overnight. After washing, 100 μl/well of biotinylated rat anti-mouseIL-2 (clone JES6-5H4), IL-4 (clone BVD6-24G2), IL-5 (clone TRFK4), IL-6(clone MP5-32C11), IL-10 (clone SXC-1) and INF-γ (clone XMG1.2)monoclonal antibodies were added and incubated for 45 min at RT. Aftersix washes, streptavidin-peroxidase conjugated was added and incubatedfor 30 min at RT. Finally, the plates were developed using ABTS.

Depletion of CD8 + Spleen Cells.

The CD8 + cell subset was depleted using MINIMACS™ (cell separator)Magnetic Ly-2 Microbeads according to the manufacturer's instructions(Miltenyi Biotec). Depleted cell preparations contained 1% CD8 + cells.

FACSCAN™ (cytometer) Analysis

Approximately 5×10⁵ cells were incubated in staining buffer (PBSsupplemented with 2% FCS and 0.1% sodium azide) with the desiredantibody or combination of antibodies for 30 min at 4° C. After washes,cells were analysed on a FACSCAN™ (cytometer) (Becton Dickinson). Themonoclonal antibodies used were FITC-conjugated anti-CD4 and anti-CD8(clones H129.19 and 53-6.7; Pharmingen).

Example 7 Cell Proliferation

Cell Proliferation Assay

Spleen cell suspensions were enriched for CD4 + T cells using MINIMACS™(cell separator) Magnetic Ly-2 and indirect goat-anti-mouse-IgGMicrobeads according to the instructions of the manufacturer (MitenyiBiotec GmbH, Germany). Cell preparations contained >65% of CD4 + cells.Cells were adjusted to 2×10⁶ cells/ml in complete medium supplementedwith 20 U/ml of mouse rIL-2 (Pharmigen), seeded at 100 μl/well in aflat-bottomed 96-well microtiter plate (Nunc, Roskilde, Denmark) andincubated for four days in the presence of different concentrations ofsoluble 1-gal. During the final 18 hours of culture 1 μCi of[³H]-thymidine (Amersham International, Amersham, U.K.) was added perwell. The cells were harvested on paper filters using a cell harvesterand the [³H]-thymidine incorporated into the DNA of proliferating cellswas determined in a .beta.-scintillation counter.

Example 8 Characterization of ssr Genes and Construction andCharacterization of the ssr Mutant S. Typhimurium Strains MvP284, MvP320and MvP333

Homology of the Two Component Regulator Genes ssrA and ssrB of SPI2 withOther Bacterial Proteins

The SPI2 gene ssrA encodes a protein similar to sensor components ofbacterial two component regulatory systems as has been described before(Ochman et al., 1996). For consistency with the nomenclature of SPI2virulence genes (Hensel et al., 1997b; Valdivia and Falkow, 1997), thisgene is designated ssrA. Downstream of ssrA, an ORF with coding capacityfor a 24.3 kDa protein was identified. This gene shares significantsimilarity with a family of genes encoding transcriptional activatorslike DegU of Bacillus subtilis, UvrY of E. coli and BvgA of Bordetellapertussis. Therefore, it is likely that the protein acts as theregulatory component of the ssr system and the gene was designated ssrB.

Inverse Regulation of SPI1 and SPI2

The expression of the type III secretions systems of SPI1 and SPI2 istightly regulated by environmental conditions. While SPI1 is inducedduring late loglearly stationary phase after growth in rich media ofhigh osmolarity and limiting O₂ (oxygen) concentration, no induction ofSPI2 gene expression was observed. In contrast, after growth in minimalmedium with limiting amounts of Mg²⁺ (8 μM) the ssaB::luc fusion washighly expressed while the sipC::lacZ fusion was not expressed. Theexpression of the ssaB::luc fusion is dependent on the function ofSsrA/B, since there is no expression in the ssrB-negative backgroundstrain P8G12 (Hensel et al., 1998). The expression of the sipC::lacZfusion is dependent on HilA, the transcriptional regulator of SPI1. Wealso observed that a mutation in ssrB affects expression of thesipC::lacZ fusion. This indicates that SPI2 has a regulatory effect onthe expression of SPI1 genes.

Bacterial strains harbouring a luc fusion to ssaB in SPI2 (strainMvP131) and a lacZ fusion to sipC in SPI1 (strain MvP239) were grownunder conditions previously shown to induce SPI gene expression.Bacteria were grown over night in minimal medium containing 8 μM Mg²⁺ orover night in LB broth containing 1% NaCl (LB 1% NaCl). The Luc activityof strain MvP131 and β-galactosidase activity of strain MvP239 weredetermined. As a control, both reporter fusions were assayed in the ssrBnegative strain background of P8G12.

Expression levels of lacZ Reporter-gene fusions to SPI genes wereassayed as described by Miller, 1992.

Construction and Analysis of sseA Reporter Gene Fusion

A 1.1 kb SmaI/HincII fragment of p5-4 was subcloned into pGP101, asuicide vector for the generation of luc fusions (Gunn and Miller,1996). The resulting construct, in which 1.0 kb upstream and 112 bp ofsseA is transcriptionally fused to luc was used to transform E. coliS17-1 λpir, and conjugational transfer to S. typhimurium performed asdescribed previously (Gunn and Miller, 1996). Strains that hadintegrated the reporter gene fusion into the chromosome by homologousrecombination were confirmed by PCR and Southern hybridization analysis.Subsequently, the fusion was moved by P22 transduction into thewild-type and various mutant strain backgrounds with mTn5 insertions inSPI1 or SPI2 genes (Maloy et al., 1996). As a control, a strain wasconstructed harbouring a chromosomal integration of pLB02, a suicideplasmid without a promoter fusion to the luc gene (Gunn and Miller,1996). For the analysis of gene expression, strains were grown for 16 hin minimal medium with aeration. Aliquots of the bacterial cultures werelysed and luciferase activity was determined using a luciferase assaykit according to the manufacturer's protocol (Boehringer Mannheim).Photon detection was performed on a Microplatescintillation/luminescence counter (Wallac, Turku). All assay were donein triplicate, and replicated on independent occasions.

Expression of sseA is Dependent on SsrAB

To establish if the sse genes are part of the SPI2 secretion system, theexpression of an sseA::luc reporter gene fusion, integrated byhomologous recombination into the chromosome of different SPI2 mutantstrains, has been investigated (FIG. 11). Transcriptional activity ofsseA in a wild-type background during growth in minimal medium wasdramatically reduced by inactivation of the SPI2 two-component system.Transposon insertions in ssrA (mutant strain P3F4) and ssrB (mutantstrain P8G12), encoding the sensor component and the transcriptionalactivator, respectively, resulted in 250 to 300-fold reduced expressionof sseA. Inactivation of hilA, the transcriptional activator of SPI1(Bajaj et al., 1996), had no effect on sseA gene expression. Transposoninsertions in two genes encoding components of the SPI2 type IIIsecretion apparatus (ssaJ::mTn5 and ssaT::mTn5; mutant strains P11D10and P9B7; Shea et al., 1996) also had no significant effect on theexpression of sseA. These data show that SsrA/B is required for theexpression of sseA, but that hilA is not.

Expression of SPI2 Genes within Macrophages is Dependent on SsrA/B

The presence of S. typhimurium within eukaryotic cells (macrophages)induces the expression of SPI2 genes as indicated by analysis of fusionsto ssaB and ssaH. This expression is dependent on the two componentregulatory system SsrA/B encoded by SPI2.

The murine macrophage-line cell line J744 was used for this experiment.Macrophages were infected at a multiplicity of infection of 10 bacteriaper macrophage with MvP131 (luc fusion to ssaB), MvP266 (luc fusion ofssaH) and MvP244 (luc fusion to ssaB in a ssrB negative background).Extracellular bacteria were killed by the addition of gentamicin (20μg/ml). At various time points, macrophages were lysed by the additionof 0.1% Triton X-100, and intracellular bacteria were enumerated byplating serial dilutions onto LB agar plates. A further aliquot of thebacteria was recovered and the luciferase activity was determined.Luciferase activities were expressed a relative light emission perbacteria.

Effects of a Mutation in ssrB on the Secreted Effector Protein of SPI1SipC

Analysis of proteins secreted into the growth medium by the S.typhimurium SPI2 mutant strain MvP320 (non-polar mutation in a, FIG. 12)revealed the absence or strong reduction in the amounts of the secretedSPI1 effector protein (Hensel et al., 1997b). These SPI2 mutants arealso reduced in their ability to invade cultured epithelial cells orcultured macrophages (Hensel et al., 1997b). To examine this phenomenonin greater detail, we expressed recombinant SipC (rSipC) and raisedantibodies against rSipC in rabbits. In Western blots, antiserum againstrSipC reacted with a 42 kDa protein from precipitates of culturesupernatants of S. typhimurium wild-type strain NCTC12023. No reactionwas observed with supernatants from cultures of EE638, a straindeficient in SipC (Hueck et al., 1995). Furthermore, in Western blotsSipC could not be detected in culture supernatants of the SPI2 mutantsMvP320. However, SipC was detected in culture supernatants of other SPI2mutants like P2D6 (ssa V::mTn5), P9B6 (ssa V::mTn5) and NPssa V(ssaV::aphT) (Deiwick et al., 1998). The detection by antiserum of SipC inculture supernatants of various strains was in accord with the presenceor absence of SipC as detected by SDS-PAGE. Further it was analyzedwhether the absence of SipC in culture supernatants of SPI2 mutantstrains was due to defective secretion of SipC via the type IIIsecretion system or reduced synthesis of SipC in these strains.Antiserum against rSipC was used to detect SipC in pellets of culturesgrown under inducing conditions for the expression of SPI1 genes (i.e.stationary phase, high osmolarity, low oxygen) (Bajaj et al., 1996).Analysis of wild-type and strains carrying various mutations in SPI1 andSPI2 genes indicated highly reduced amounts of SipC in the mutants witha non-polar mutation in ssrB. However, SipC was detected at levelscomparable to those observed in pellets of wild-type cultures and SPI2mutant strains P2D6, P9B6 and NPssa V. The effect on SipC synthesis isnot due to reduced growth rates or reduced protein levels in SPI2mutants, since both parameters were comparable for the wild-type andSPI2 mutants.

Effects of a Mutation in the SPI2 Gene ssrB on the Expression of SPI1Genes

In order to assay the effect of SPI2 mutations on the expression of SPI1genes, previously characterized fusions of lacZ to various SPI1 genes(Bajaj et al., 1995; Bajaj et al., 1996) were transduced into the SPI2mutant MvP320 and various SPI1 mutants to generate a set of reporterfusion strains. The expression of the reporter β-galactosidase incultures grown under conditions inducing for SPI1 expression (see above)was assayed. A Tn insertion in hilA (P4H2) reduced the expression ofprgK as well as sipC, while an insertion in spaRS(P6E11) only affectedthe expression of sipC. Some mutant strains with a mutation in the SPI2gene ssrB encoding a components of the two component regulatory systemshowed reduced expression of reporter fusions to prgK and sipC (FIG.11). The effects on the expression of both genes was similar. Othermutant strains with Tn insertions in ssaV (P2D6, P9B6), as well asmutant NPssaV harbouring a non-polar insertions in ssaV, had levels ofexpression of prgK and sipC comparable to that of corresponding reporterfusions in a wild-type genetic background. Analysis of lacZ fusions toprgH and invF revealed a similar effect on expression as shown for prgKand sipC.

A Mutation in the SPI2 Gene ssrB Affects Expression of the SPI1Regulator hilA

Analysis of reporter fusions to sipC and prgK indicated that expressionof genes in two different operons of SPI1 can be affected by SPI2mutations, suggesting that these mutations affect other SPI1 genesinvolved in regulation of sipC and prgK. It has been demonstratedpreviously that the expression of SPI1 genes is under the control of thetranscriptional activator HilA (Bajaj et al., 1995; Bajaj et al., 1996).The expression of hilA was therefore analyzed in the presence of a SPI2mutation in ssrB. The SPI2 mutant strain MvP320 had largely diminishedlevels of hilA expression. Again, very low levels of hilA expressionwere observed in mutants that had reduced levels of prgK and sipCexpression. To analyze whether the effect of the SPI2 mutation on sipCexpression resulted from the reduced expression of hilA, we nextperformed complementation experiments in various mutant strainsharbouring pVV135 (constitutive expression of hilA) (Bajaj et al., 1996)or pVV214 (expression of hilA from the native promoter) (Bajaj et al.,1995). In accordance with a previous study (Bajaj et al., 1995), thehilA mutation of strain P4H2 was complemented by pVV214. However, thesipC expression was not restored in the mutant strain MvP320 harbouringeither pVV135 or pVV214.

Construction of the ssrA and ssrB Mutant S. typhimurium Strains MvP284and MvP320

-   -   Mutant MvP284, ssrA. The ssrA gene (FIG. 12) was subcloned from        the phage clone λ2 derived plasmid p2-2 on a 5.7 kb BamHI        fragment in pUC18 as indicated in Table 1. A 1.6 kb fragment was        recovered after HindIII and EcoRV digestion of p2-2 and        subcloned in HindIII/HincII-digested pbluescript II KS +. The        resulting construct termed p2-20 was digested with HincII and        dephosphorylated with alkaline phosphatase. The aphT cassette        was isolated as described above and ligated to the linearized        plasmid p2-20 in the same orientation into the unique HincII        site. After transformation of E. coli XL-1 Blue and selection        against kanamycin and carbenicillin (50 μg/ml each) one clone        has been chosen and the harbouring plasmid isolated. This        plasmid was termed p2-21 and its identity proved via restriction        analysis. p2-21 was further digested with KpnI and XbaI, a 2.5        kb fragment isolated and ligated to KpnI/XbaI-digested pKAS32.        This plasmid was electroporated into E. coli CC118 λpir and        transformants selected to kanamycin and carbenicillin (50 μg/ml        each). As done before, one clone was chosen, its plasmid with        the according DNA fragment in pKAS32, termed p2-22, isolated and        confirmed by restriction analysis. Plasmid p2-22 was        electroporated into E. coli S17-1 λpir and transferred into S.        typhimurium NCTC12023 (streptomycin resistant) by conjugation as        has been described previously (de Lorenzo and Timmis, 1994).        Exconjugants in which the ssrA gene had been replaced by the        cloned gene disrupted by insertion of the aphT cassette were        selected by its growth on M9 + glucose minimal medium agar        plates (Maloy et al., 1996) and its resistance to kanamycin and        carbenicillin (100 μg/ml). The resulting exconjugants were        finally shown to have a lactose negative phenotype and to be        sensitive to kanamycin and streptomycin. Selected clones were        further examined by Southernblot analysis. In order to exclude        possible mutations which might have been developed during the        cloning procedure the mutated ssrA allele was transfered into a        fresh Salmonella background by P22 transduction (described by        Maloy et al., 1996). The resulting Salmonella strain MvP284 was        examined for the presence of the resistance cassette within the        ssrA gene by the use of primers ssrA-For (5′-AAG GAA TTC AAC AGG        CAA CTG GAG G-3′) (SEQ ID NO:64) and ssrA-Rev (5-CTG CCC TCG CGA        AAA TTA AGA TAA TA-3′) (SEQ ID NO65). Amplification of DNA from        clones containing the wild-type ssrA allele resulted in a PCR        product of 2800 bp, use of DNA from clones harbouring a ssrA        allele disrupted by the aphT cassette resulted in a PCR product        of 3750 bp. The resulting Salmonella strain MvP320 was examined        for the presence of the resistance cassette within the ssrB gene        by the use of Southern hybridization analysis of total DNA of        exconjugants.    -   Mutant MvP320, ssrB. The ssrB gene (FIG. 12) was subcloned from        the phage clone λ1 derived plasmid p1-6 on a 4.8 kb        PstI/BamHI-fragment in pT7-Blue as indicated in Table 1. A 1.7        kb fragment was recovered after BamHI and HincII digestion of        p1-6 and subcloned in BamHI/HincII-digested pBluescript II KS +.        The resulting construct termed p1-20 was digested with EcoRV and        dephosphorylated with alkaline phosphatase. The aphT cassette        was isolated as described above and ligated to the linearized        plasmid p1-20 in the same orientation into the unique EcoRV        site. After transformation of E. coli XL-1 Blue and selection        against kanamycin and carbenicillin (50 μg/ml each) one clone        has been chosen and the harbouring plasmid isolated. This        plasmid was termed p1-21 and its identity confirmed by        restriction analysis. p1-21 was further digested with KpnI and        XbaI, a 2.5 kb fragment isolated and ligated to        KpnI/XbaI-digested pKAS32. This plasmid was electroporated        into E. coli CC118 λpir and transformed bacteria selected to        kanamycin and carbenicillin (50 μg/ml each) was performed. As        done before, one clone was chosen, its plasmid with the        according DNA fragment in pKAS32, termed p1-22, isolated and        confirmed by restriction analysis. Plasmid p1-22 was        electroporated into E. coli S17-1 λpir and transferred into S.        typhimurium NCTC12023 (streptomycin resistant) by conjugation as        has been described previously (de Lorenzo and Timmis, 1994).        Exconjugants in which the ssrB gene had been replaced by the        cloned gene disrupted by insertion of the aphT cassette were        selected by its growth on M9 + glucose minimal medium agar        plates (Maloy et al., 1996) and its resistance to kanamycin and        carbenicillin (100 μg/ml). The resulting exconjugants were        finally shown to have a lactose negative phenotype and to be        sensitive to kanamycin and streptomycin. Selected clones were        further examined by Southernblot analysis. In order to exclude        possible mutations which might have been acquired during the        cloning procedure the mutated ssrB allele has been transferred        into a fresh Salmonella background by P22 transduction        (described by Maloy et al., 1996). Screening of mutants with a        insertion of the aphT cassette within the ssrB locus was        performed by PCR using primers ssrB-For (5′-CTT AAT TTT CGC GAG        GG-3′) (SEQ ID NO:66) and ssrB-Rev (5′-GGA CGC CCC TGG TTA        ATA-3′) (SEQ ID NO:67). Amplification of DNA from clones        containing the wild-type ssrB allele resulted in a PCR product        of 660 bp, use of DNA from clones harbouring a ssrB allele        disrupted by insertion of the aphT cassette resulted in a PCR        product of 1600 bp. The resulting Salmonella strain MvP320 was        examined for the presence of the resistance cassette within the        ssrB gene by the use of Southern hybridization analysis of total        DNA of exconjugants.        Construction of the Mutant Strain MvP340 Carrying an In-frame        Deletion in ssrA

A deletion of 407 codons between codon 44 and 451 of ssrB was generated.Plasmid p2-2 was digested by BamHI and KpnI, a fragment of 3.7 kb wasrecovered and subcloned in pBluescript KS + to generate p2-50. Plasmidp2-50 was linearized by digestion with PstI, which cuts once within thesubcloned fragment of the ssrA gene. Primers ssrA-del-1 (5′-GGT CTG CAGGAT TTT TCA CGC ATC GCG TC-3′) (SEQ ID NO: 68) and ssrB-del-2 (5′-GGTCTG CAG AAC CAT TGA TAT ATA AGC TGC-3′) (SEQ ID NO:69) were designed tointroduce PstI sites. PCR was performed using linearized p2-50 astemplate DNA. The TAQPLUS® polymerase (Pyrococcus furiosus DNApolymerase+recombinant T. Aquaticus YT1 polymerase) (Stratagene) wasused according to the instructions of the manufacturer. Reactions of 100μl volume were set up using 10 μl of 10× TAQPLUS® Precision buffercontaining magnesium chloride, 0.8 μl of 100 mM dNTPs, 250 ng DNAtemplate (linearized p2-50), 250 ng of each primer and 5 U of TAQPLUS®DNA polymerase (Pyrococcus furiosus DNA polymerase+recombinant T.Aquaticus YT1 polymerase). PCR was carried out for 35 cycles of: 95° C.for 1 minute, 60° C. for 1 minute, 72° C. for 6 minutes. Then a finalstep of 72° C. for 10 minutes was added. 10 μl of the PCR reaction wereanalyzed. A product of the expected size was recovered, digested byPstI, self-ligated, and the ligation mixture was used to transform E.coli DH5á to resistance to carbenicillin. Plasmids were isolated fromtransformants and the integrity of the insert and the deletion wasanalyzed by restriction analysis and DNA sequencing. The insert of aconfirmed construct was isolated after digestion with XbaI and KpnI andligated to XbaI/KpnI-digested vector pKAS32. The resulting construct wasused to transform E. coli S17-1 λpir to resistance to carbenicillin, andconjugational transfer of the plasmid to S. typhimurium (Nal^(R),Strep^(R)) was performed according to standard procedures (de Lorenzoand Timmis, 1994). Exconjugants that had integrated the suicide plasmidby homologous recombination were selected by resistance to nalidixicacid and carbenicillin, and screened for sensitivity to streptomycin.Such clones were grown in LB to OD600 of about 0.5 and aliquots wereplated on LB containing 250 μg/ml streptomycin to select for colonieswhich had lost the integrated plasmid and undergone allelic exchange.Clones resistant to streptomycin but sensitive to carbenicillin wereused for further analysis. Screening of mutants with a deletion withinthe ssrA locus was performed by PCR using primers ssrA-For (5′-AAG GAATTC AAC AGG CAA CTG GAG G-3′) (SEQ ID NO:64) and ssrA-Rev (5-CTG CCC TCGCGA AAA TTA AGA TAA TA-3′) (SEQ ID NO:65). Amplification of DNA fromclones containing the wild-type ssrA allele resulted in a PCR product of2800 bp, use of DNA from clones harbouring a ssrA allele with aninternal deletion resulted in a PCR product of 1580 bp. The integrity ofclones harbouring the ssrA deletion was further confirmed bySouthernanalysis of the ssrA locus. Finally, the ssrA locus containingthe internal in-frame deletion was moved into a fresh strain backgroundof S. typhimurium by P22 transduction (Maloy et al., 1996) and theresulting strain was designated MvP340.

Southern Hybridization

Genomic DNA of Salmonella was prepared as previously described (Henselet al., 1997). For Southern hybridization analysis, genomic DNA wasdigested with EcoRI or EcoRV, fractionated on 0.6% agarose gels andtransferred to Hybond N⁺ membranes (Amersham, Braunschweig). Variousprobes corresponding to the ssrA and ssrB region were obtained asrestriction fragments of the subcloned insert of λ1 and λ2.

Example 9 Evaluation of Safety of S. Typhimurium Strain MvP320

For competition assays between S. typhimurium wild-type and the mutantstrain MvP320, bacteria were grown in LB to an optical density at 600 nmof 0.4-0.6. Cultures were diluted and aliquots of the two cultures weremixed to form an inoculum containing equal amounts of both strains. Theratio of both strains was determined by plating dilutions on LB platescontaining antibiotics selective for individual strains. An inoculum ofabout 10⁴ colony forming units (cfu) was used to infect 6 to 8 weeks oldfemale BALB/c mice (Charles River Breeders, Wiga) by injection into theperitoneal cavity. At several time points after infection mice weresacrificed by cervical dislocation and the bacterial load of liver andspleen was determined by plating tissue homogenates using the ‘WASP’(Meintrup, Lähden) spiral plating device. Plating was performed using LBplates containing 50 μg/ml kanamycin or 100 μg/ml nalidixic acid toselect for the mutant strains or the wild-type, respectively.

Strain MvP320 harbouring the aphT gene cassette in ssrB was recovered inat least 1000-fold lower numbers than the S. typhimurium wild-typestrain. These data indicate that ssrB contributes significantly tosystemic infections of S. typhimurium in the mouse model ofsalmonellosis.

Statistical Analysis of all Experiments.

Statistical significance between paired samples was determined byStudent's t test. The significance of the obtained results wasdetermined using the statgraphic plus for windows 2.0 software(Statistical Graphic Corp.).

Example 10 Characterization of the in vivo Inducible P_(ssaE) Promoter(Promoter B, FIG. 24B)

The promoter which is located upstream of ssaE (P_(ssaE), formerlycalled Promoter B) was shown to be regulated by the ssrAB locus. A DNAfragment comprising nucleotide 800 to 120 (800-1205) in the includedsequence (FIG. 21A) was shown to confer ssrB-dependent regulation uponthe expression of a reporter gene (gfp) fused to the promoter. The DNAfragment was cloned on a low-copy plasmid in front of the gfp gene. Ashas been shown previously for other reporter gene constructs, inductionof expression from P_(ssaE) (800-1205) was observed in magnesium minimalmedium (Deiwick et al., 1999) and was dependent on the presence of achromosomal wild type allele of ssrB. A shorter DNA fragment, comprisingnucleotide 923 to 1205 (923-1205) in the included sequence, did notconfer regulation upon expression of gfp. However, expression wasreduced compared to the P_(ssaE) (800-1205) fragment and was not inducedin magnesium minimal medium nor was it dependent on ssrB. Thus, theP_(ssaE) (800-1205) fragment comprises promoter active and regulatorysequences, probably including an SsrB-binding site.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Map of Salmonella Pathogenicity Island 2 (A) indicating thepositions of the mutations in strains MvP101, MvP102, and MvP103 (B). Apartial restriction map of the genomic region is shown, and thepositions of plasmid inserts relevant for this work are indicated (C).B, BamHI; C, ClaI; E, EcoRI; P, PstI; V, EcoRV; S, SmaI; EMBL databaseaccession numbers are indicated for the sequences in (A).

FIG. 2 a. Alignment of the deduced SseB amino acid sequence (SEQ IDNO:5) to EspA of EPEC (SEQ ID NO:40) (Elliot et al., 1998). The ClustalWalgorithm of the MacVector 6.0 program was used to construct thealignments. Similar amino acid residues are boxed, identical residuesare boxed and shaded.

FIG. 2 b. Alignment of the deduced SseC amino acid sequence (SEQ IDNO:8) to EspD of EPEC (SEQ ID NO:41) (Elliot et al., 1998), YopB ofYersinia enterocolitica (SEQ ID NO:42) (Hakansson et al., 1993), andPepB of Pseudomonas aeruinosa (SEQ ID NO:43) (Hauser et al., 1998). TheClustalW algorithm of the MacVector 6.0 program was used to constructthe alignments. Positions where at least three amino acid residues aresimilar are boxed, where at least three residues are identical are boxedand shaded.

FIG. 3. Intracellular accumulation of S. typhimurium SPI2 mutants in RAW264.7 macrophages. Following opsonization and infection, macrophageswere lysed and cultured for enumeration of intracellular bacteria(gentamicin protected) at 2 h and 16 h post-infection. The values shownrepresent the fold increase calculated as a ratio of the intracellularbacteria between 2 h and 16 h post-infection. Infection was performed inin triplicates for each strain and the standard error from the mean isshown.

FIG. 4. Intracellular survival and replication of SPI2 mutant S.typhimurium in (A) J774.1 cells and (B) periodate-elicited peritonealmacrophages from C3H/HeN mice. After opsonization and internalization,phagocytes were lysed and cultured for enumeration of viableintracellular bacteria at time 0 h. The values shown represent theproportion of this intracellular inoculum viable at 20 h+the standarderror of the mean. Samples were processed in triplicate, and eachexperiment was performed at least twice.

FIG. 5. β-gal-specific antibodies in intestinal lavages of mice orallyimmunized with either MvP101[pAH97], MvP103 [pAH97], SL7207 [pAH97] orMvP101 at day 52 after immunization. Results are expressed as percentageof the corresponding total Ig subclass present in the intestinal lavage,the SEM is indicated by vertical lines. Significant levels ofantigen-specific IgM could not be detected in any of the groups. Theresults obtained with MvP103 and SL7207 (not shown) were similar tothose for MvP101.

FIG. 6. β-gal-specific proliferative response of CD4 + enriched spleencells from mice orally immunized with either MvP101 [pAH97], MvP103[pAH97], SL7207 [pAH97] or MvP101. Cells were restimulated in vitroduring a 4 day incubation with different concentrations of solubleβ-gal. The values are expressed as mean cpm of triplicates; the SEM wasin all cases lower than 10%. Background values obtained from wellswithout the stimulating antigen were subtracted. Results obtained withMvP103 and SL7207 (not shown) were similar to those obtained withMvP101.

FIG. 7. IFN-γ present in supernatants from cultured CD4 + enrichedspleen cells of mice orally immunized with either MvP101 [pAH97], MvP103[PAH97], SL7207 [pAH97] or plasmidless MvP101 at day 2 and 4 of culture.Spleen cells were isolated from mice at day 52 after immunization, andCD4 + enriched populations were restimulated in vitro for four days inthe presence of soluble β-gal (20 μg/ml). IFN-γ production wasdetermined by ELISA, results represent the means of threedeterminations. The SEM is indicated by vertical lines, similar resultswere obtained using any of the plasmidless carriers (not shown). Nosignificant differences with the control groups were observed when IL-2,IL-4, IL-5, IL-6 and IL-10 were tested (not shown).

FIG. 8. Kinetics of the β-gal-specific serum IgG (closed symbols) andIgM (open symbols) antibody responses in mice (n=5) after oralimmunization with either MvP101 [pAH97] (triangle), MvP103 [pAH97](circle), SL7207 [pAH97] (square) or plasmidless MvP101 (diamond).Results are expressed as the reciprocal log₂ of the geometric mean endpoint titer (GMT), the SEM was in all cases lower than 10%. Similarresults were obtained using any of the plasmidless carriers (not shown),immunizations are indicated by arrows.

FIG. 9. Subclass profiles of the β-gal-specific IgG antibodies presentin the serum of mice (n=5) orally immunized with either MvP101 [pAH97],MvP103 [pAH97], SL7207 [PAH97] or plasmidless MvP101 at day 52post-immunization. Results are expressed as ng/ml, the SEM is indicatedby vertical lines. Similar results were obtained using any of theplasmidless carriers (not shown).

FIG. 10. Recognition of the MHC class I-restricted βGP1 epitope bylymphocytes primed in vivo in mice by oral vaccination with eitherMvP101 [pAH97], MvP103 [pAH97], SL7207 [pAH97] or plasmidless MvP101.Spleen cells from immunized mice were restimulated in vitro five days inthe presence of 20 μM βGP1. At the end of the culture, lymphocytes weretested in a [³H]-thymidine-release assay using P815 (open symbols) andβGP1-loaded P815 (closed symbols) as targets. Results are mean values oftriplicate wells (one out of three independent experiments is shown) andare expressed as: [(retained cpm in the absence ofeffectors)−(experimentally retained cpm in the presence ofeffectors)/retained cpm in the absence of effectors]×100; SEM were lowerthan 5% of the values. Similar results were obtained using any of theplasmidless carriers (not shown).

FIG. 11. Expression of an sseA::luc fusion in wild-type and mTn5 mutantstrains of S. typhimurium.

FIG. 12. Map of Salmonella Pathogenicity Island 2 (A) indicating thepositions of the mutations in strains MvP284 and MvP320 (B). A partialrestriction map of the genomic region is shown, and the position ofinserts of plasmids relevant for this work is indicated (C). B, BamHI;C, ClaI; H, HindIII; P, PstI; V; S, SmaI; EcoRV; II, HincII.

FIG. 13. Model for the transcriptional organization of SPI2 virulencegenes. This model is based on the observation of the transcriptionaldirection of SPI2 genes, characterization of promoter activities

FIG. 14 shows the principle of how mutations having a different grade ofattenuation can be generated. As shown in A, the inactivation of oneeffector gene such as sse results in a low grade of attenuation. Asshown in B, the additional inactivation of a gene located outside theSPI2 locus such as aroA results in a medium grade of attenuation. Byinsertional mutation with a polar effect all genes in a polycistroniccluster are affected which results in a high grade of attenuation, asshown in C. As shown in D, the inactivation of a regulatory gene such asssrB results in a supreme attenuation.

FIG. 15 shows the principle of insertional mutation by example ofinsertional mutation into a virulence gene. Different cassettes such asSMC, GEC, TC and/or invertase cassette may be inserted into a clonedvirulence gene, thus yielding an inactivated virulence gene which may beintroduced into a cell by homologous recombination using a virulencegene cassette.

FIG. 16 illustrates the selective marker cassette.

FIG. 17 illustrates the gene expression cassette and the inductionthereof in a two-phase system. The gene expression cassette comprises apromoter, optionally a gene cassette comprising one or more expressionunits and optionally one or more transcriptional terminators for theexpression units and/or a transcribed sequence 5′ to the gene expressioncassette.

FIG. 18 shows the structural requirements of the gene expression unitfor the delivery of heterologous antigens into various compartments,i.e. accessory sequences that direct the targeting of the expressionproduct.

FIG. 19 shows a transactivator cassette in a one-phase system and atwo-phase system.

FIG. 20 shows different modes of gene expression as realized by thecombination of different accessory sequences and/or cassettes in aone-phase system and a two-phase system.

FIG. 21A shows the genomic sequence of a region of the SPI2 locus fromSalmonella comprising the complete sequences of the genes ssaE to ssaIand partial sequences of ssaD and ssaJ (cf. FIG. 12) (SEQ ID NO:1).

FIG. 21B shows the nucleotide sequence of a region of the SPI2 locusfrom Salmonella comprising the coding sequences for ssrA and ssrB (SEQID NO:2).

FIGS. 22A-Q each show the nucleotide sequence of the respective geneindicated (SEQ ID NOS:3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, and 36).

FIGS. 23A-Q each show the amino acid sequence of the respectivepolypeptide indicated. (SEQ ID NO:4, 6, 8, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, and 37)

FIGS. 24A,B each show a nucleotide sequence comprising an in vivoinducible promoter (SEQ ID NO:38 and SEQ ID NO:39).

Literature

Bajaj, V., Lucas, R. L., Hwang, C., Lee, C. A. (1996). Co-ordinateregulation of Salmonella typhimurium invasion genes by environmental andregulatory factors is mediated by control of expression. Mol. Microbiol.22: 703-714.

Bajaj, V., Hwang, C., Lee, C. A. (1995). hilA is a novel ompR/toxRfamily member that activates the expression of Salmonella typhimuriuminvasion genes. Mol. Microbiol. 18: 715-727.

Baudry, B., Kaczorek, M. and Sansonetti, P. J. (1988) Nucleotidesequence of the invasion plasmid antigen B and C genes (ipaB and ipaC)of Shigella flexneri. Microb Pathog 4: 345-357.

Bogdanove, A. J., Beer, S. V., Bonas, U., Boucher, C. A., Collmer, A.,Coplin, D. L., Cornelis, G. R., Huang, H.-C., Hutcheson, S. W.,Panopoulos, N. J., and van Gijsegem, F. (1996) Unified nomenclature forbroadly conserved hrp genes of phytopathogenic bacteria. Mol Microbiol20: 681-683.

Buchmeier, N. A., Lipps, C. J., So, M. Y. H., Heffron, F. (1993)Recombination-deficient mutants of Salmonella typhimurium are avirulentand sensitive to the oxidative burst of macrophages. Mol. Microbiol. 7:933-936

Chang, A. C. and Cohen, S. N. (1978). Construction and characterizationof amplifiable multicopy DNA cloning vehicles derived from the P15Acryptic miniplasmid. J Bacteriol 134: 1141-1156.

Cirillo, D. M., Valdivia, R. H., Monack, D. M., Falkow, S. (1998)Macrophage-dependent induction of the Salmonella pathogenicity island 2type III secretion system and its role in intracellular survival. Mol.Microbiol. 30: 175-88

Cirillo, J. D., Stover, C. K., Bloom, B. R., Jacobs, W. R. Jr.,Barletta, R. G. (1995). Bacterial vaccine vectors and bacillusCalmette-Guerin. Clin. Infect. Dis. 20: 1001-1009.

DeGroote, M. A., Ochsner, U. A., Shiloh, M. U., Nathan, C., McCord, J.M., Dinauer, M. C., Libby, S. J., Vazquez-Torres, A., Xu, Y., and Fang,F. C. (1997) Periplasmic superoxide dismutase protects Salmonella fromproducts of phagocyte NADPH-oxidase and nitric oxide synthase. Proc NatlAcad Sci USA 94: 13997-14001.

de Lorenzo, V. and Timmis, K. N. (1994). Analysis and construction ofstable phenotypes in Gram-negative bacteria with Tn5-and Tn10-derivedminitransposons. Methods Enzymol. 235, 386-405.

Devereux, J., Haeberli, P., and Smithies, O. (1984) A comprehensive setof sequence analysis programs for the VAX. Nucleic Acids Res 12:387-395.

Deiwick, J., Nikolaus, T., Erdogan, S., Hensel, M. (1999). Environmentalregulation of Salmonella pathogenicity island 2 gene expression. Mol.Microbiol. 31: 1759-1773.

Deiwick, J., Nikolaus, T., Shea, J. E., Gleeson, C., Holden, D. W.,Hensel, M. (1998). Mutations in SPI2 genes affecting transcription ofSPI1 genes and resistance to anti-microbial agents. J. Bacteriol. 180:4775-4780.

Donnenberg, M. S., and Kaper, J. B. (1991) Construction of an eaedeletion mutant of enteropathogenic Escherichia coli using apositive-selection suizide vector. Infect Immun 59: 4310-4317

Elliot, S. J., Wainwright, L. A., McDaniel, T. K., Jarvis, K. G., Deng,Y. K., Lai., L. C., McNamara, B. P., Donnenberg, M. S., and Kaper, J. B.(1998) The complete sequence of the locus of enterocyte affacement (LEE)from enteropathogenic Escherichia coli E2348/69. Mol Microbiol 28: 1-4.

Fields, P. I., Swanson, R. V., Haidaris., C. G., and Heffron, F. (1986)Mutants of Salmonella typhimurium that cannot survive within themacrophage are avirulent. Proc Natl Acad Sci USA 83: 5189-5193.

Forsberg, A., Pavitt, G. D. and Higgins, C. F. (1994). Use oftranscriptional fusions to monitor gene expression: a cautionary tale.J. Bacteriol. 176: 2128-2132.

Galán, J. E., Ginocchio, C. and Costeas, P. (1992). Molecular andfunctional characterization of the Salmonella invasion gene invA:homology of InvA to members of a new protein family. J. Bacteriol. 174:4338-4349.

Gentschev, I., Glaser, I., Goebel, W., McKeever, D. J., Musoke, A., andHeussler, V. T. (1998) Delivery of the p67 sporozoite antigen ofTheileria parva by using recombinant Salmonella dublin: secretion of theproduct enhances specific antibody responses in cattle. Infect Immun 66:2060-2064.

Gunn, J. S., Miller, S. 1. (1996). PhoP-PhoQ activates transcription ofpmrAB, encoding a two-component regulatory system involved in Salmonellatyphimurium antimicrobial peptide resistance. J. Bacteriol. 178:6857-6864.

Hakansson, S., Schesser, K., Persson, C., Galyov, E. E., Rosqvist, R.,Homble, F., and Wolf Watz, H. (1996) The YopB protein of Yersiniapseudotuberculosis is essential for the translocation of Yop effectorproteins across the target cell plasma membrane and displays acontact-dependent membrane disrupting activity. EMBO J 15: 5812-5823.

Harlow, E., Lane, D. (1988). Antibodies: A laboratory manual. ColdSpring Harbour Laboratory Press, Cold Sprin Harbour, New York.

Hauser, A. R., Fleiszig, S., Kang, P. J., Mostov, K., and Engel, J. N.(1998) Defects in type III secretion correlate with internalization ofPseudomonas aeruginosa by epithelial cells. Infect Immun 66: 1413-1420.

Heithoff, D. M., Sinsheimer, R. L., Low, D. A., and Mahan, M. J. (1999)An essential role for DNA adenine methylation in bacterial virulence.Science 284: 967-970.

Hensel, M., Nikolaus, T., Egelseer, C. (1999). Molecular and functionalanalysis indicates a mosaic structure of Salmonella Pathogenicity Island2. Mol. Microbiol. 31: 489-98.

Hensel, M., Shea, J. E., Baumler, A. J., Gleeson, C., and Holden, D. W.(1997a) Analysis of the boundaries of Salmonella pathogenicity island 2and the corresponding chromosomal region of Escherichia coli K-12. JBacteriol 179: 1105-1111.

Hensel, M., Shea, J. E., Raupach, B., Monack, D., Falkow, S., Gleeson,C., and Holden, D. W. (1997b) Functional analysis of ssaJ and the ssaK/Uoperon, 13 genes encoding components of the type III secretion apparatusof Salmonella pathogenicity island 2. Mol Microbiol 24: 155-167.

Hensel, M., Shea, J. E., Gleeson, C., Jones, M. D., Dalton, E., Holden,D. W. (1995). Simultaneous identification of bacterial virulence genesby negative selection. Science 269: 400-403.

Herrero, M., de Lorenzo, V., and Timmis, K. N. (1990) Transposon vectorscontaining non-antibiotic resistance selection markers for cloning andstable chromosomal insertion of foreign genes in gram-negative bacteria.J Bacteriol 172: 6557-6567.

Hess, J., Dietrich, G., Gentschev, I., Miko, D., Goebel, W., andKaufmann, S. H. (1997a) Protection against murine listeriosis by anattenuated recombinant Salmonella typhimurium vaccine strain thatsecretes the naturally somatic antigen superoxide dismutase. InfectImmun 65: 1286-1292.

Hess, J., Gentschev, I., Miko, D., Weizel, M., Ladel, C., Goebel, W.,and Kaufmann, S. H. (1996) Superior efficacy of secreted over somaticantigen display in recombinant Salmonella vaccine induced protectionagainst listeriosis. Proc Natl Acad Sci USA 93: 1458-1463.

Hess, J., Miko, D., Gentschev, I., Dietrich, G., Goebel, W., Mollenkopf,H. J., Ladel, C., and Kaufmann, S. H. (1997b) Modulation of antigendisplay by attenuated Salmonella typhimurium strains and its impact onprotective immunity against listeriosis. Behring Inst Mitt 160-171.

Hofmann, K. and Stoffel, W. (1993). TMbase—a database of membranespanning proteins segments. Biol Chem Hoppe-Seyler 347: 166.

Holtel, A., Timmis, K. N., Ramos, J. L. (1992%. Upstream bindingsequences of the XylR activator protein and integration host factor inthe xylS gene promoter region of the Pseudomonas TOL plasmid. Nucleic.Acids. Res. 20:1755-1762.

Hueck, C. J. (1998). Type III protein secretion systems in bacterialpathogens of animals and plants. Microbiol. Mol. Biol. Rev. 62: 379-433.

Hueck, C. J., Hantman, M. J., Bajaj, V., Johnston, C., Lee, C. A.,Miller, S. I. (1995). Salmonella typhimurium secreted invasiondeterminants are homologous to Shigella Ipa proteins. Mol. Microbiol.18: 479-490.

Kaniga, K., Tucker, S., Trollinger, D., Galan, J. E. (1995). Homologs ofthe Shigella IpaB and IpaC invasins are required for Salmonellatyphimurium entry into cultured epithelial cells. J. Bacteriol. 177:3965-3971.

Kuwajima, G., Kawagishi, I., Homma, M., Asaka, J., Kondo, E., andMacnab, R. M. (1989) Export of an N-terminal fragment of Escherichiacoli flagellin by a flagellum-specific pathway. Proc Natl Acad Sci USA86: 4953-4957.

Laemmli, U. K. (1970). Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 227: 680-685.

Macnab, R. M. (1996) Flagella and motility. In Escherichia coli andSalmonella: cellular and molecular biology. F. C. Neidhardt, et al.(eds.). Washington, D.C.: ASM Press, pp. 123-145

Maloy, S. R., Steward, V. L. and Taylor, R. K. (1996). Genetic analysisof pathogenic bacteria, Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press.

Miller, J. H. (1992). A short course in bacteria genetics. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.

Miller, S. I., Kukral, A. M., and Mekalanos, J. J. (1989) A twocomponent regulatory system (phoP and phoQ) controls Salmonellatyphimurium virulence. Proc Natl Acad Sci USA 86: 5054-5058.

Miller, V. L., Mekalanos, J. J. (1988). A novel suicide vector and itsuse in construction of invertion mutations: osmoregulation of outermembrane proteins and virulence determinants in Vibrio cholerae requirestoxR. J. Bacteriol. 170, 2575-2583.

Minamino, T., and Macnab, R. M. (1999) Components of the Salmonellaflagellar export apparatus and classification of export substrates. JBacteriol 181: 1388-1394.

Monack, D. M., Raupach, B., Hromockyj, A. E., and Falkow, S. (1996)Salmonella typhimurium invasion induces apoptosis in infectedmacrophages. Proc Natl Acad Sci USA 93: 9833-9838.

O'Callaghan, D., Charbit, A. (1990). High efficiency transformation ofSalmonella typhimurium and Salmonella typhi by electroporation. Mol.Gen. Genet. 223:156-158.

Ochman, H., Soncini, F. C., Solomon, F., Groisman, E. A. (1996).Identification of a pathogenicity island required for Salmonellasurvival in host cells. Proc. Nat. Acad. Sci. USA 93: 7800-7804.

Orr, N., Galen, J. E., and Levine, M. M. (1999) Expression andimmunogenicity of a mutant diphtheria toxin molecule, CRM₁₉₇, and itsfragments in Salmonella typhi vaccine strain CVD 908-htrA. Infect Immun67: 4290-4294.

Pallen, M. J., Dougan, G., and Frankel, G. (1997) Coiled-coil domains inproteins secreted by type III secretion systems. Mol Microbiol 25:423-425.

Ralph, P., Prichard, J., and Cohn, M. (1975). Reticulum cell sarcoma:and effector cell in antibody-dependent cell-mediated immunity. J.Immunol. 114: 898-905.

Reed, L. J., Muench, H. (1938). A simple method of estimating fiftypercent end points. Am. J. Hyg. 27:493-497.

Sambrook, J., Fritsch, E. F., Maniatis, T. (1989). Molecular cloning: alaboratory manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.

Sanger, F., Nicklen, S., and Coulson, A. R. (1977) DNA sequencing withchain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463-5467.

Schmitt, C. K., Darnell, S. C., and O'Brien, A. D. (1996) The attenuatedphenotype of a Salmonella typhimurium flgM mutant is related toexpression of FliC flagellin. J Bacteriol 178: 2911-2915

Shea, J. E., Hensel, M., Gleeson, C., Holden, D. W. (1996).Identification of a virulence locus encoding a second type III secretionsystem in Salmonella typhimurium. Proc. Natl. Acad. Sci. USA 93:2593-2597.

Skorupski, K. and Taylor, R. K. (1996). Positive selection vectors forallelic exchange. Gene 169: 47-52.

Valdivia, R. H., and Falkow, S. (1997) Fluorescence-based isolation ofbacterial genes expressed within host cells. Science 277: 2007-2011.

Valentine, P. J., Devore, B. P., and Heffron, F. (1998) Identificationof three highly attenuated Salmonella typhimurium mutants that are moreimmunogenic and protective in mice than a prototypical aroA mutant.Infect Immun 66: 3378-3383

Wattiau, P., Bernier, B., Deslée, P., Michiels, T., and Cornelis, G. R.(1994) Individual chaperones required for Yop secretion by Yersinia.Proc Natl Acad Sci USA 91: 10493-10497.

Yanisch-Perron, C., Vieira, J. and Messing, J. (1985). Gene 33, 103-119.

Young, G. M., Schmiel, D. H., and Miller, V. L. (1999) A new pathway forthe secretion of virulence factors by bacteria: the flagellar exportapparatus functions as a protein-secretion system. Proc Natl Acad SciUSA 96: 6456-6461.

Zhu, N., Liggitt, D., Liu, Y., Debs, R. (1993). Systemic gene expressionafter intravenous DNA delivery into adult mice. Science 261: 209-211.

1. An isolated attenuated gram-negative Salmonella cell comprising theSPI2 gene locus, wherein at least one effector (sse) gene selected fromthe group consisting of sseC, sseD and sseE of the SPI2 locus isinactivated, wherein said inactivation results in anattenuation/reduction of virulence compared to the wild type of saidcell.
 2. The isolated attenuated gram-negative Salmonella cell accordingto claim 1, wherein said cell has a broad host range.
 3. The isolatedattenuated gram-negative Salmonella cell according to claim 2, whereinsaid cell is a Salmonella serotype Typhimurium Definitive Type 104(DT104) cell.
 4. The isolated attenuated gram-negative Salmonella cellaccording to claim 1, wherein at least one gene is inactivated by amutation comprising a deletion.
 5. The isolated attenuated gram-negativeSalmonella cell according to claim 4, wherein said deletion comprises atleast 6 nucleotides.
 6. The isolated attenuated gram-negative Salmonellacell according to claim 4, wherein the mutation comprises a deletion ofthe complete coding sequence for said gene.
 7. The isolated attenuatedgram-negative Salmonella cell according to claim 1, wherein at least onegene is inactivated by a mutation comprising the insertion of aheterologous nucleic acid molecule.
 8. The isolated attenuatedgram-negative Salmonella cell according to claim 7, wherein saidmutation is a non-polar mutation.
 9. The isolated attenuatedgram-negative Salmonella cell according to claim 1, wherein at least oneadditional gene located outside of the SPI2 locus is inactivated,wherein the inactivation results in a further attenuation/reduction ofvirulence compared to the wild type.
 10. The isolated attenuatedgram-negative Salmonella cell according to claim 9, wherein saidadditional gene is superoxide dismutase.
 11. The isolated attenuatedgram-negative Salmonella cell according to claim 1, comprising at leastone selective marker cassette.
 12. The isolated attenuated gram-negativeSalmonella cell according to claim 11, wherein said selective markercassette is capable of conferring an antibiotic resistance to the cell.13. The isolated attenuated gram-negative Salmonella cell according toclaim 1 comprising at least one gene expression cassette.
 14. Theisolated attenuated gram-negative Salmonella cell according to claim 1comprising at least one transactivator cassette.
 15. The isolatedattenuated gram-negative Salmonella cell according to claim 1 comprisingat least one invertase cassette.
 16. The isolated attenuatedgram-negative Salmonella cell according to claim 1 further comprising atleast one insertion cassette.